Comprehensive Bioactive Natural Products, Volume 2: Efficacy, Safety & Chlinical Evaluation (Part I)

Comprehensive Bioactive Natural Products Volume 2

Efficacy, Safety & Clinical Evaluation I

V.K. GUPTA Indian Institute of Integrative Medicine (Council of Scientific & Industrial Research) Canal Road, Jammu (J&K State)- 180 001, India

2010

CID

Studium Press LLC, U.S.A.

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Comprehensive Bioactive Natural Products

VoL 2: Efficacy, Safety & Clinical Evaluation I © Copyright 2010

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the editor and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. All rights are reserved under International and Pan-American Copyright Conventions. Apart from any fair dealing for the purpose of private study, research, criticism or review, as permitted under the Copyright Act, 1956, no part of this publication may be reproduced, stored in a retrieval system or transmitted, in any form or by any means-electronic, electrical, chemical, mechanical, optical, photocopying, recording or otherwise-without the prior permission of the copyright owner.

ISBN: 1-933699-52-3 SERIES ISBN: 1-933699-50-7

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STUDIUM PRESS, LLC P.O. Box-722200, Houston, Texas-77072, USA Tel. 713-541-9400; Fax:713-541-9401 E-mail: [email protected]

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Comprehensive Bioactive Natural Products: (Multi-Volume Set) Series Editor: V.K. Gupta E-mail: [email protected];[email protected] Volumes Published (2010) VoL 1: Potential & Challenges Ed. V.K Gupta Vol. 2: Efficacy, Safety & Clinical Evaluation I Ed. V.K Gupta Vol. 3: Efficacy, Safety & Clinical Evaluation II Ed. V.K Gupta Vol. 4: Antioxidants & Nutraceuticals Eds. V.K Gupta & Anil K Verma Vol. 5: Immune-modulation & Vaccine Adjuvants Ed. V.K Gupta Vol. 6: Extraction, Isolation & Characterization Eds. V.K Gupta, S.C. Taneja & B.D. Gupta Vol. 7: Structural Modifications & Drug Development Eds. V.K Gupta, S.C. Taneja & B.D. Gupta Vol. 8: Quality Control & Standardization Eds. V.K. Gupta, S.C. Taneja & B.D. Gupta

MEMBERS Dr. A. Panossian: Swedish Herbal Institute Research and Development, Kovlingevagen 21, Vallberga, SE-312 50, Sweden; E-mail: [email protected] Prof. Yu Zhao: Department ofTCM & Natural Drug Research, College of Pharmaceutical Sciences, Room 513, Zhejiang University, Zijingang Campus, 388 Yuhangtang Rd., Hangzhou 310058, China; Email: dryuzhao®Zju.edu.cn;[email protected] Prof. A. Evidente: Department of Organic Chemistry, Dipartimento di Scienze del Suolo, della Pianta, dell'Ambiente e delle Produzioni Animali, Universita di Napoli Federico II, Portici, Italy; E-mail: [email protected] Prof. Mirian Salvador: Instituto de Biotecnologia, Universidade de Caxias do SuI, Rua Francisco Getulio Vargas, 1130, CEP 95070-560, Caxias do SuI, Rio Grande do SuI, Brazil; E-mail: [email protected]

Dr. Gregory Beck: Department of Biology, University of Massachusetts Boston, 100 Morrissey Blvd., Boston, MA 02125-3393, 617-287-6619, 6684; E-mail: [email protected] Dr. Stephen M. Sagar: Departments of Oncology and Medicine, McMaster University, Hamilton, Ontario, Radiation Oncologist, Juravinski Regional Cancer Centre, Hamilton Health Sciences Centre. Associate Brain-Body Institute (St Josephs Health Care Centre and McMaster University); E-mail: [email protected] Dr. Anil K. Verma: Sr. Asstt. Professor of Zoology, Govt. (P.G.) College for Women, Gandhi Nagar, Jammu-180 001(J&K State), India; E-mail: [email protected] Prof. Ian Fraser Pryme: Dept. of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway; E-mail: [email protected] Dr. Robert Frangez: Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Slovenia; E-mail: [email protected] Dr. George Qian Li: Herbal Medicines Research and Education Centre Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia, E-mail: [email protected] Prof. Yuji Nagashima: Department of Food Sciences and Technology, Tokyo University of Marine Sciences and Technology, Konan 4-5-7, Minato, Tokyo 108-8477, Japan; E-mail: [email protected] Prof. Pius Mpiana Tshimankinda: Departement de Chimie, Faculte des Sciences, Universite de Kinshasa, B.P. 190, Kinshasa XI, RD Congo; E-mail: [email protected] Prof. Supayang Piyawan Voravuthikunchai: Natural Products Research Center and Department of Microbiology, Faculty of Science, Prince of Songkla University, Hatyai, Songkla, Thailand - 90112; E-mail: [email protected]

SERIES ISBN: 1-933699-50-7

About the Series Nature abounds with a rich potential heritage ofbioactive natural products as that have been exploited for effective and beneficial use either as a therapeutic resource against a human disease or as a chemopreventive, antioxidant, nutraceutical or diet supplement. A natural product is a substance or a chemical compound produced by a living organism found in nature that usually has a pharmacological or biological activity. Some of these agents are derived from terrestrial plants, whereas, others are obtained from microorganisms, marine or freshwater organisms and even the higher animals. Past few decades have witnessed an unprecedented worldwide growth in the interest of bioactive natural products based medicines, both in developed and developing countries, and offer a resource-pool for lead identification in drug discovery owing to the reason that isolation of a novel molecule is easier than de novo synthesis, especially when the molecular structure of the compound is very complex. On account of such advantages, pharmaceutical companies are opting for bioactive natural products based drugs. More than 20 new drugs, launched world over between 2000 and 2005, originate from natural products. Scrutiny of medical indications by source of compounds has demonstrated that natural products and related drugs are used to treat 87% of all categorized human diseases (infectious and non-infectious). The development of new bioassay techniques, biotechnology methods, screening and high performance analytical methods have introduced new concepts and possibilities of rational drug design and drug discovery. Interest in bioactive natural products research is growing more strongly and can be attributed to several factors, including remarkable diversity of natural products, unmet therapeutic needs, the development of novel and sensitive techniques to detect, isolate, purify and structurally characterize these active constituents. This has opened opportunities for multidisciplinary research that joins the forces of natural product chemistry, molecular and cellular biology, synthetic and analytical chemistry, bio-chemistry, biotechnology, general biology, and pharmacology to exploit the vast diversity of bioactive natural products. Taking cognizance of these facts, we have initiated efforts in editing the present series "Comprehensive Bioactive Natural Products" and brought eight volumes (1 to 8) providing edited information from over 139 original research and review articles by eminent scientists and researchers

from India and abroad, representing 40 countries ofthe world on wide range oftopics in the area of natural products categorized under the themes viz., 1. Potential & Challenges 2. Efficacy, Safety & Clinical Evaluation I 3. Efficacy, Safety & Clinical Evaluation II 4. Antioxidants & Nutraceuticals 5. Immune-modulation & Vaccine Adjuvants 6. Extraction, Isolation & Characterization 7. Structural Modifications & Drug Development 8. Quality Control & Standardization

These volumes critically evaluate the present state-of-art, current status and future prospects in a well-illustrated manner. It is hoped that this series, which reflects the contributor's research results as well as world wide reviews will be widely read and used by all interested in these fields and shall open new vistas of research and academic pursuits for those engaged in the fields of bioactive natural products. I would like to take this opportunity to thank each of the authors who have contributed in the preparation of this book series.

Jammu, India V.K. Gupta Series Editor

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AMITY INSTITUTE FOR HERBAL AND BIOTECH PRODUCTS DEVELOPMENT - An Institution of Ritnand Balved Education Foundation - Thiruvananthapuram

Prof. (Dr.) P. PUSHPANGADAN, M.Sc. M.Phil. Ph.D., FBRS FES. FNRS, FNSE, FNESA, FNAASc, FNASc., (UN Equator Initiative Laureate) Director General & Senior Vice President, RBEF (Former Director, NBRI, Lucknow)

08-06-2009

Foreword to the Series Medicine among the ancient civilizations was a blend of Religion and Science. Natural products played an important role throughout the world in the prevention and treatment of diseases. These natural products originated from varied sources such as plants, animals, micro-organisms, marine organisms, terrestrial vertebrates and invertebrates. In man's quest for discovering new and potent remedies for diseases, he keenly observed his immediate neighbourhood and experimented not only with organic substances but also with in-organic materials such as metals, minerals, ores etc. Over the years, the accumulated medical wisdom was compiled into excellent treatises and these have later developed into the various classical systems of medicine associated with the ancient civilizations and cultures of the world. Apart from the classical systems of medicines, which by its nature and practice had universal applications, there existed another stream of medicine in the countryside, among the less privileged class of people, among the forest dwellers and among the nomadic tribes which were location specific, confined to small communities, tribes and families. These medical systems were mostly perpetuated by oral traditions. Nevertheless, the value of the oral traditions cannot be overlooked as these localized knowledge systems are veritable treasures of invaluable information on the properties and uses of hundreds of plant, animal and mineral products. Ethno medical information is now increasingly being utilized in the search for novel bioactive molecules from natural products. Several well known plants such as Licorice (Glycyrrhiza glabra) myrrh (Commiphora sps) and poppy capsule latex (Papaver somniferum) were mentioned in the clay tablets dating back to 2600 B.C, obtained from Mesopotamia. These plants and their derivatives are still used in various forms as herbal drugs as well as pure isolates in different systems of medicine. For instance,

codeine, papaverine, morphine etc isolated from P. somniferum are still used in modem medicine. Similarly hemisuccinate carbenoxolone sodium, a semi synthetic derivative of glycyrrhetic acid isolated from licorice is used for the treatment of gastric and duodenal ulcers. Plants, especially those with ethno medical history have been the major source ofbio active molecules. According to a study by Fabricant and Farnsworth (2001), out of 122 plant derived drugs, 80% had their origin from plants with ethno medical use. Since secondary metabolites from plants are products ofbiosynthetic reactions occurring in living systems, bioactive molecules from natural sources are excellent drug candidates as they are perceived to show more biological friendliness than synthetic drugs. The ever increasing popularity and acceptance of natural products have evinced an extra ordinary interest and scope in the subject in recent years. Compendium of Bioactive Natural Products- a series of eight books edited by Dr. V.K Gupta (Volumes 1,2,3 and 5) and Dr. V.K Gupta, Dr. S.C. Taneja, Dr. B.D. Gupta and Dr. A.K.Verma (Volumes 4,6,7 and 8), published by MIS. Studium press LLC, U.S.A are an excellent compilation, giving comprehensive and reliable data on the current development in the field ofbioactive natural products. This series contain 139 original research and review articles contributed by a galaxy of eminent scientists and academicians from 40 countries around the world. The editors have taken care to invite eminent scholars in the relevant areas to contribute each chapter in the series. The series deals with wide ranging topics related to natural products such as extraction, isolation and characterization, structural modifications and drug development, quality control and standardization and, immune modulation and vaccine adjuvants, antioxidants and nutraceuticals, efficacy, safety and clinical evaluation (Parts-1 and 2) and potentials and challenges. The editors of this series Dr. V.K Gupta, Dr. S.C. Taneja, Dr. B.D. Gupta and Dr. Anil K Verma are known to me for more than three decades and they are all stalwarts in natural products and related areas. It is gratifying to know that this series is dedicated to the memory of Col. Sir Ram Nath Chopra, the Founder Director of Regional Research Laboratory, now known as Indian Institute of Integrative Medicine (IIIM), Jammu.

Col. Sir R.N. Chopra (1882-1973) was a pioneer of systematic studies of indigenous drugs, promoter of Indian Systems of Medicine and Patron of Pharmacy in India. Most of the medicinal plants, he studied were in use, in Ayurveda for thousands of years. The major fields of Cot. Chopra's research were general pharmacology, chemotherapy, indigenous drugs, drug addiction and drug assays. At the Calcutta school of Tropical Medicine, he developed a pattern of research in studying the action of medicinal plants which influenced an entire generation of pharmacologists. Pharmacists and physiologists in India. In the words of Dr. G.V. Satyawati, Former Director General ofIndian Council of Medical Research, the credit of kindling the interest of Indian Chemists and Pharmacologists in Medicinal Plants should rightfully go to

Sir Ram Nath Chopra who, has been claimed as the Father of Indian Pharmacology' . I had the privilege and good fortune of meeting Col, Sir. Ram Nath Chopra along with Prof. Dr. E.K. Janaky Aromal at his residence in 1969 immediately after my joining Regional Research Laboratory as a Scientific Assistant. My meeting with Col. Sir R.N. Chopra had left a lasting impression of a great scientist in my memory. It is a fitting tribute to Col. Sir. R.N. Chopra that the editors have decided to dedicate these eight volumes to his memory. I congratulate the editors for bringing out this series which is timely, relevant and important to all those who are engaged in the study of natural products and development of therapeutically useful bioactive molecules. I am sanguine that these volumes will receive wider acceptance among the scientific community, especially among those engaged in Natural Product research.

(P. Pushpangadan)

About the Editor Dr. Vijay Kumar Gupta, Ph.D., FLS, London (born 1953-) Deputy Director & Head, Animal House, Indian Institute ofIntegrative Medicine (CSIR), Jammu, India. He did his M.Sc. (1975) and Ph.D. (1979) in Zoology both from University of Jammu, Jammu-India. His research capabilities are substantiated by his excellent work on histopathology, ecology and reproductive biology of fishes, turtles, birds and mammals, which has already got recognition in India and abroad. Dr. Gupta has to his credit more than 75 scientific publications and review articles which have appeared in internationally recognized Indian and foreign journals. Founder fellow, life member and office bearer of many national societies, academies and associations. He has successfully completed a number of research/consultancy projects funded by various governments, private and multinational agencies. His current areas of interest are histopathology, toxicology, pre-clinical safety pharmacology and reproductive efficacy studies oflaboratory animals. He is also Editor-in-chief of the books 1) Utilisation and Management of Medicinal Plants 2) Medicinal Plants: Phytochemistry, Pharmacology and Therapeutics 3) Perspectives in Animal Ecology and Reproduction (Vols.1-6). The Editor-in-chief of the American Biographical Institute, USA, has appointed him as Consulting Editor of The Contemporary Who's Who. Dr. Gupta also appointed as Nominee for the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Govt. oflndia). Recently the Linnaean Society ofLondon, U.K. has awarded fellowship to him in November 2009 in recognition of his contribution towards the cultivation of knowledge in Science of Natural History.

Preface The natural products have been of great help to mankind in resolving many diseases from earliest time. Primitive people in all ages have had some knowledge of natural medicines. The knowledge of the primitive man has been so modified with the advancement of civilization that know-a-days almost every type of medicine be that of Ayurvedic, Unani or even a number of allopathic medicines are obtained from natural sources. The medicinal value of drug plants is due to the presence of some chemical compounds in the plant tissue that produce a definite physiological action on human body. Early societies learned through trial and error that many plants contained substances with significant curative properties and over the years the natural products became used in more refined ways leading ultimately to the use of pure single component "active ingredient" as drugs. Nature has continuously provided mankind with a broad and structurally diverse array of pharmacologically active compounds that have proved to be indispensable for the cure of deadly diseases or as lead structures for novel pharmaceuticals. Their dominant role is evident in the approximately 60% of anticancer compounds and 75% of drugs for infectious diseases that are either natural products or natural product derivatives. At present 25% of the modern medicines are developed from plants that were first used traditionally, and many synthetic drugs have also been obtained from natural precursors. Almost, 70% modern medicines in India are derived from natural products. In the USA, the process of synthetic drug discovery and development takes an average of 12 years, and any new drug requires the investment of an average of US$ 230 million. It is seen that natural drugs take a comparatively much less time and expenses than synthetic drugs. Hence natural medicines would be cheaper, unless the market price are inflated by other considerations. Despite this success, during the past couple of decades, research into natural products has experienced a steady global decline. The introduction of high-throughput synthesis and combinatorial chemistry with their promise of a seemingly inexhaustible supply of compound libraries has greatly contributed to this declining interest in the screening of natural products by the pharmaceutical industry. Resistance of the parasites to existing drugs and their higher cost warrants the search of newer drug molecules. The origin of many effective drugs is found in the traditional medicine practices and in view of this several researchers have undertaken studies to clinically evaluate the medicinal plants for their preclinical safety and efficacy. The natural product market has been continually growing at an increased rate mainly as a nutraceuticals, dietary supplements, cosmoceuticals, chemopreventives and as herbal remedies. These therapies should emphasis on well-controlled and randomized clinical trials to prove

safety and efficacy and necessary guidelines should be followed in order to harmonies the use of bioactive natural products. At present, there is limited data on safety, efficacy and clinical trials to prescribe natural products in therapy. Realizing the need to document this knowledge gained through recent researchers, the present volume "Efficacy, Safety & Clinical Evaluation-I" of the book series entitled, "Comprehensive Bioactive Natural Products" presents 23 chapters discussing therapeutic potential of a large number of bioactive natural products of plant/animal origin investigated worldwide. Some of the interesting chapters included in this volume are: Perspectives in efficacy, safety and clinical evaluation of bioactive natural products; The modern application of traditional Chinese medicine: indigo naturalis as an alternative therapy in psoriasis vulgaris; Development of anti-angiogenesis therapies for treating cancer from Chinese medicinal herbs; Inflammation and mediators; Recommendation for reporting randomized controlled trials of herbal interventions: CONSORT for herbal medicine trials; Anxiolytic activity screening studies on extracts of a few medicinal plants; Larvicidal and antimicrobial activities of seeds ofAnnona cornifolia; Antidepressant activity of the extracts of three Hypericum species native to China; Cytolytic and toxic effects of ostreolysin, a protein from the oyster mushroom CPleurotus ostreatus); Antioxidant, antisecretory and gastroprotective activities from Leiothrix fiavescens; Evaluation of the immunity potency and security of inactivated oil-adjuvant vaccine against chlamydiosis in dairy cattle; Pharmacokinetics of active constituents of Rhodiola rosea SHR-5 extract; Metabolism and pharmacokinetic studies of coumarins; N aringin and its aglycone, research and managements; Biotransformation of drugs; Green tea: molecular targets in glucose uptake, inflammation, and insulin signaling pathways; Euphorbious plants as molluscicides and piscicides. The studies included are likely to lead further researches in this direction and it is hoped that this publication would attract world wide audience of researchers and the academicians of allied disciplines engaged in the search of new drug from natural resources.

Jammu, India

V.K.Gupta

ISBN: 1-933699-52-3

Table of Contents About the Series Foreword to the Series Preface Table of Contents of Volume 2 1.

Perspectives in Efficacy, Safety and Clinical Evaluation of Bioactive Natural Products

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1-29

YOGENDRANAYAK, VEERESHP. VEERAPUR, ANANTHA NAIK N AGAPPA AND M.K UNNIKRISHNAN (INDIA)

2.

The Modern Application of Traditional Chinese Medicine: Indigo N aturalis as an Alternative Therapy in Psoriasis vulgaris

31-43

YIN-Ku LIN AND JONG-HWEI Su PANG (TAIWAN)

3.

Development of Anti-Angiogenesis Therapies for Treating Cancer from Chinese Medicinal Herbs

45-65

STEPHEN M. SAGAR, RAIMOND K WONG AND DONALD R. YANCE JR. (CANADA, OREGON)

4.

An Anthology of the Studies on the Anti-Inflammatory, Antinociceptive and Febrifuge Effects of Medicinal Plants in Turkey

67-119

ESRA KUPEU AKKOL AND ERDEM YESILADA (TuRKEy)

5.

Comparative Study of the Antigenotoxic Effects of Some Selected Natural Plant Products Against Methylmethane Sulphonate Induced Genotoxic Damage in Cultured Mammalian Cells

121-131

YASIR HASAN SIDDIQUE AND MOHAMMAD AFZAL (INDIA)

6.

Inflammation and Mediators

A. KOUL, R. SHARMA, G.D. SINGH, A. KHAJuRIA AND V.K GUPTA (INDIA)

S. SINGH, P. KoUL,

133-178

7.

Recommendations for Reporting Randomized Controlled Trials of Herbal Interventions: CONSORT for Herbal Medicine Trials JOELJ. GAGNIER N.D., HEATHER BOON, PAULA ROCHON, DAVID MOHER, JOANNE BARNES AND CLAIRE BOMBARDIER FOR THE CONSORT GROUP (CANADA)

179-209

8.

Anxiolytic Activity Screening Studies on Extracts of a Few Medicinal Plants RICHA SHRI, MANJEET SINGH AND ANUPAM SHARMA (INDIA)

211-218

9.

Study of Eugeniajambolana and Momordica charantia in Respect to Safety and Antidiabetic Effect In vivo and In vitro YELE, SANTOSH AND VEERANJANEYULU, A. (INDIA)

219-230

10. Larvicidal and Antimicrobial Activities of Seeds of Annona cornifolia A. St. -Hil. (Annonaceae)

231-237

LUCIANA ALVES R.S. LIMA, MARIA AMELIA D. BOAVENTURA, JACQUELINE A. TAKAHASHI AND LUCIA P.S. PIMENTA (BRAZIL) 11. Antidepressant Activity of the Extracts of Three Hypericum species Native to China DEPO YANG, JIE BAr, ZHEN LI, GEORGE Q LI AND DONGMEI WANG (CHINA, AUSTRALIA)

239-249

12. Cytolytic and Toxic Effects of Ostreolysin, a Protein from the Oyster Mushroom (Pleurotus ostreatus)

251-264

KRIsTINA SEPCIC AND ROBERT FRANGE (REPUBLIC OF SLOVENIA) 13. Antioxidant, Antisecretory and Gastroprotective Activities from Leiothrix flavescens

265-279

THIAGO DE MELLO MORAEs, MARCELO APARECIDO SILVA, CLENILSON MARTINS RODRIGUES, LoURDES CAMPANER DOS SANTOS, MIRIAM SANNOMIYA, LUCIA REGINA MACHADO DA ROCHA, ALBA REGINA MONTEIRO SOUZA BRITO, TArS M. BAUAB, WAGNER VILEGAS AND CLELIAAKrKO HIRUMA-LIMA (BRAZIL) 14. Evaluation of the Immunity Potency and Security of Inactivated Oil-Adjuvant Vaccine against Chlamydiosis in Dairy Cattle Qru C., ZHOU J., CAO X. AND LIN G. (CHINA)

281-291

15. Annona crassiflora Wood Constituents: Antimalarial Efficacy, Larvicidal and Antimicrobial Activity

293-305

MARy ANE GONQALVES, VANESSA CARLA FURTADO MOSQUEIRA

AND

LUCIA PINHEIRO SANTOS PIMENTA (BRAZIL)

16. Pharmacokinetics of Active Constituents of Rhodiola rosea SHR-5 Extract A PANOSSIAN, A HOVHANNISYAN, H. ABRAHAMYAN, E. GABRIELYAN AND G. WIKMAN (ARMENIA, SWEDEN)

307-329

17. Metabolism and Pharmacokinetic Studies of Coumarins BEHERA D. AND AnDEPALLI V. (INDIA)

331-353

18. N aringin and its Aglycone, Research and Managements RICKYW.K WONG AND FANNYY.F. YOUNG (HONG KONG)

355-387

19. Biotransformation of Drugs M.G. TYAGI, S. PRABHAKARAN AND KR. DHANASEKHAR (INDIA)

389-398

20. Pharmacokinetics and Drug Interactions of Glycyrrhizin/Glycyrrhiza AK AGGARWAL AND D.K MAJuMDAR (INDIA)

399-416

21. Determination of Total Antioxidant Status and Rosmarinic Acid from Orthosiphon stamineus in Rat Plasma GABRIEL AKYIREM AKOWUAH AND ISMAIL ZHARI (MALAYSIA)

417-427

22. Green Tea: Molecular Targets in Glucose Uptake, Inflammation, and Insulin Signaling Pathways HEPING CAO, ANNE M. ROUSSEL AND RICHARD A ANDERSON (USA, FRANCE)

429-448

23. Euphorbious Plants as Molluscicides and Piscicides: A Review RAM P. YADAV AND AJAY SINGH (INDIA) Appendix

449-460

Table of Contents of Other Volumes of the SeriesVois. 1 & 3 to 8

461-474

Index

475-493

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1 Perspectives in Efficacy, Safety and Clinical Evaluation of Bioactive Natural Products YOGENDRA NAYAK1, VEERESH P. VEERAPUR2 , ANANTHA NAIK NAGAPPA3 AND M.K UNNIKRISHNAN 1

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ABSTRACT Natural products (NPs) include crude drugs, extracts, botanicals and pure compounds obtained from nature. NPs offer a rich source of diverse biologically active compounds with high molecular diversity, varying structural complexity and unparalleled novelty. A significant portion of the currently prescribed drugs owe their origin either directly or indirectly to NPs. The bioactivity of NPs not only builds the foundation for ethnic medicine systems of the world but also provides an impetus to modern drug discovery. Isolation of active component / molecule, structure elucidation, biological evaluation, dereplication, semisynthesis, biosynthesis, as well as optimization of downstream processing have undergone revolutionary changes in recent times, thereby enhancing the rate of drug discovery. The advent of high throughput screening and the availability of combinatorial compound libraries in natural products have been attracting major innovators towards natural products. Translational research has brought the drug discovery team closer to the clinical setting and has simultaneously increased the collective involvement of clinicians and pharmaceutical scientists. Complexity of natural products is fitly matched by the complexity 1. Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal- 576 104, India. 2. Department of Pharmaceutical Chemistry, SET's College of Pharmacy, S.R. Nagar, Dharwad - 580 002, India. 3. Department of Pharmaceutical Management, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal- 576 104, India. * Corresponding author : E-mail: [email protected]

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of several pathological conditions, for instance diabetes, where multiple drug targets work in unison for producing a therapeutic outcome. Reverse pharmacology makes use of clinical evidences from traditional medicines, and then working backwards towards validation of claims in the laboratory. All the above themes are discussed in detail. Key words: Combinatorial compound library, drug discovery, high throughput screening, multiple drug targets, natural products, reverse pharmacology, translational research

IMPACT OF NATURAL PRODUCTS ON DRUG DISCOVERY: HISTORICAL PERSPECTIVES Natural products are rich sources of biologically active compounds with tremendous molecular diversity, possessing great promise in drug discovery and development (Butler, 2004; Newman et al., 2003a; Koehn et al., 2005; 2004b; Chin et al., 2006; Cragg et al., 2006; Blunt et al., 2007; Gordaliza, 2007; Butler et al., 2008). There is no dearth of illustrative examples to prove this paradigm. One of the oldest drugs, aspirin has had its origin from a plant (bark of willow), making it a historical land mark (Vane, 2000; Robert-II et al., 2001). Penicillin was first isolated from a microorganism Penicillum notatum (Wainwright, 1990). Morphine obtained from opium poppy is still an important drug in pain management. Bergmann's discovery and subsequent identification of spongothymidine and spongouridine in the early 1950s from the Caribbean sponge Tethya crypta led to the development of a nucleoside with biological activity (Bergmann, 1950). An explosive growth in synthetic chemistry further led to the discovery of a close analogue, cytosine arabinoside, a potent antileukemic agent (Beranek, 1986; Suckling, 1991). This compound was subsequently commercialized by Upjohn (now Pharmacia) as Ara-C. A closely related compound adenine arabinoside (Ara-A) was later synthesized and commercialized by Burroughs Wellcome (now Glaxo Smith Kline). Similarly, insulin was first isolated from the pancreatic extracts of animals such as cows, pigs and sheep, till recombinant DNA technology replaced the old technology. Eli Lilly (Indianapolis, USA) later demonstrated the commercial feasibility of recombinant DNA technology with the introduction of human insulin in 1982, ushering in a paradigm shift in drug discovery and development, leading to the start of the 'biotech era' (Pillai, 2001). The discovery of cyclosporine from microbial sources revolutionized immunosuppressive therapy for organ transplant patients (Kahan, 1999). Indeed, it has been estimated that at least 60% of the roughly 1184 new therapeutic agents introduced world-wide over the last twenty five years (1981-2006) have had their origins from natural products in one way or the other (Newman et al., 2007). The changing strategies and innovations in NP research have greatly influenced drug discovery programmes, in which

Efficacy, Safety and Clinical Evaluation of Bioactive Natural Products

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sample selection and collection, processing, isolation of active componenti molecule, structure elucidation, biological evaluation, dereplication, semisynthesis, biosynthesis, as well as optimization of downstream processing has become the norm. Such strategies have substantially enhanced the rate of discovery of novel NP with renewed interest in recent years (Gullo et al., 2005; Lawrence, 2005a, 2007b). With the advance of high throughput screening (HTS) and new instrumentation, the discovery of the new molecules with biological activity has hastened further. Newer guidelines and statutes implemented by various regulatory authorities have enhanced the time frame for the assessment of safety and efficacy of drugs in human populations. It can take up to fifteen years for the development of a molecule before it is commercialized into a marketed drug. Apart from the passage of time, a great amount of money is being spent in the process of developing a product into a clinically useful drug (Frank, 2003; Schmid et al., 2007). It is therefore important to examine the various events in drug discovery from natural sources and the associated regulatory rigors in assessment of quality, efficacy and safety of the drug. It is also essential to look into the translational aspects of NPs from preclinical to clinical studies (Dickson et al., 2004). The recent increase in the development-costs of new chemical entities (NCEs) has forced many innovators to search for active drug molecules from nature. The vast reservoir of diverse sources and the infinite variety in nature's molecular repertoire offers a fascinating arena for the drug discovery team.

NATURAL PRODUCTS FROM PLANT ORIGIN Medicines from plant sources have been used since ancient times for a variety of ailments. Herbal preparations have been used in the art of healing since time immemorial. Primitive man, through trial and error, gained knowledge of herbals and passed it on to their progeny. It appears that for thousands of years herbs were perhaps used for both their magical powers as well as medicinal values (Oumeish, 1998). Ancient civilization flourished in 3000 BC onward in Egypt, Middle East, India and China, with a parallel growth in the refinement of herbal medicine. The Egyptian Ebers Papyrus (1500 BC) is one of the earliest records providing information on herbs (Frey, 1986; EI-Gammal, 1994). In India, circa 1500 BC, Vedas included the Ayurveda system of medicine along with its vastly sophisticated informationbased on herbs, with about 350 herbals cited in this compilation (Patwardhan et al., 2004). Chinese traditional pharmacopoeia lists over 5700 traditional medicines which are mostly of herbal origin (Qiu, 2007). Chinese traditional medicine now enjoys credibility that is comparable to Western medicine in many parts of the world. Many universities in China teach and practice herbal medicine. Over the last 10 years Europe has witnessed a growth in the practice of Traditional Chinese Herbal Medicine.

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The development of organic chemistry has shown that the ability of herbal medicine to treat the body depended upon its chemical constituents. Chemists first began extracting and isolating chemicals from plants in the 18 th century. Encouraging results accelerated their enterprise and NP chemists have given to the world some of its most useful drugs such as morphine, digoxin, d-tubocurarine, ephedrine, quinine, vincristine, paclitaxel etc. A detailed list is presented in Table 1. Table 1. Prescription drugs derived from natural sources

Use Amino glycoside antibiotic

Analgesic

Drug name and the source Streptomycin (Bacteria; Streptomyces griseus) Gentamicin (Micromonospora) Kanamycin (Streptomyces kanamyceticus) Neomycin (Streptomyces fradiae) Netilimicin sulfate (Micromonospora) Morphine (Fruit exudate; Papaver somniferum)

Non-opioid analgesic

Epibatidine (Alkaloid that originally is found in the skin of a neotropical poisonous frog, Epipedobates tricolor); 200 times stronger than morphine; derivatives are under clinical investigation

Ansamycin antibiotic;

Rifamycin (Amycolatopsis rifamycinica); Anti-TB

Anti-Alzheimer's

Galantamine hydrobromide (An alkaloid obtained from Galanthus nivalis); Selective acetylcholinesterase inhibitor

Antibiotic

Mupirocin (Mixture of several pseudomonic acids obtained from Pseudomonas fluorescens); active against methicillin-resistant Staphylococcus aureus (MRSA) Chloramphenicol (bacterium; Streptomyces venezuelae) Tetracycline (polyketide antibiotic from Streptomyces) Daptomycin (Streptomyces roseosporus); Cyclic lipopeptide antibacterial agent for skin infections Teicoplanin (Actinoplanes teichomyceticus); Glycopeptide antiobiotic active against MRSA Vancomycin (Amycolatopsis orientalis); Glycopeptide antibiotic active against penicillin resistant Staphylococcus aureus Lincomycin (Streptomyces lincolnensis); Lincosamide antibiotic Erythromycin (actinomycete, Saccharopolyspora erythraea); Macrolide antibiotic Aztreonam (Chromobacterium violaceum); Monobactam antibiotic Cephalosporin (fungus; Cephalosporium acremonium); Beta lactum antibiotic Penicillin (Fungus; Penicillum notatum); Beta lactum antibiotic Colistin (polymyxin E) (Bacillus polymyxa var. colistinus); Polymyxin antibiotic Bacitracin (Bacillus subtilis var Tracy); Polypeptide antibiotic Fusidic acid (fungus: Fusidium coccineum); Urinary antibiotic

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Table 1. Contd. Use

Drug name and the source

Anticancer

Arglabin (from a species of wormwood Artemisia glabella endemic to the Karaganda region of Kazakstan) Bleomycin (Glycopeptide antibiotic from bacterium: Streptomyces vertic ill us) Etoposide (Herb: podophylum). Masoprocol (Nordihydroguaiaretic acid; NDGA; is a potent antioxidant compound found in the long-lived creosote bush: Larrea tridentata) Paclitaxel (plant; Taxus brevifolia) Solamargines (plant; Solanum marginatum) Vinblastin, vincristine (herb; Vinca rosea)

Anticancer antibiotic

Daunorubicin (Streptomyces peucetius) Pentostatin (Streptomyces antibioticus) Daunomycin (from bacterium: Streptomyces peucetius) Doxorubicin (Adriamycin; related to daunomycin from new strain of Streptomyces peucetius) Mithramycin (Plicamycin; Streptomyces plicatus) Mitomycin C (Streptomyces lavendulae) Sarkomycin (Streptomyces erythrochromogenes).

Anticoagulant

Heparin (sheep liver)

Antidiabetic

Insulin (pig and sheep) Voglibose (Aspergillus awamori)

Antifertility

Diosgenin (rhizome; Dioscorea deltoideae)

Antihypertensive

Reserpine (Rauwolfia serpentina)

Antimalaria

Quinine (Cinchona bark) Artemisinin (herb, Artemisia Annua)

Antiplatelet

Bivalirudin anew, genetically engineered form of hirudin, the substance in the saliva of the leech (Haementeria officinalis); used to reduce the risk of blood clotting in unstable angina who are undergoing a procedure to open blocked arteries in the heart.

Anti-spasmodic

Ephedrine (herb: Ephedra)

Cardiotonic

Digoxin (Fox glove; Digitalis species)

Hypolipidemic

Mevastatin (fungus; Penicillium citrinum)

Immunosuppressant for organ transplant

Sirolimus (Rapamycin; from bacterium Streptomyces hygroscopicus) Tacrolimus (bacterium Streptomyces tsukubaensis) Cyclosprin (fungus; Tolypocladium inflatum)

Local anaesthetic

Cocaine (plant; Erythroxylum coca)

Muscle relaxant

d-tubocurarine (plant; Chondrodendron tomentosum)

Mydriatic

Atropine (herb; Atropa belladonna)

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In spite of the great advances in synthetic and combinatorial chemistry, medicinal plants still make an important contribution in pharmaceutical innovation. According to the World Health Organization (WHO), about 65-80% of the world's population living in developing countries, still depends essentially on plants for primary health care because of poverty and lack of access to modern medicine (Pal et al., 2003). Some Western countries such as Germany, France, Italy and the United States have developed appropriate guidelines for registration of herbal medicines. Discovery of the blockbuster anticancer drug paclitaxel (Taxol) from the yew tree (Taxus brevifolia) once again helped to revive the interest towards herbal medicine (McChesney et al., 2007). Nowadays, several major pharmaceutical companies demonstrate renewed interest in investigating plants for discovering new sources for new lead structures and also for the development of standardized phytotherapeutic agents with improved efficacy, safety and quality (Gordaliza, 2007; Potterat et al., 2008).

NATURAL PRODUCTS FROM MARINE SOURCES Marine NPs have yielded a considerable number of new drug candidates which are still in preclinical or early clinical development (Haefner, 2003; Marris, 2006; Saleem et al., 2007). However a few are already in the market, such as cytarabine. Drugs from marine sources have a wide range of therapeutic benefits such as anticancer, antiviral, antiinfective, antimicrobial, analgesic, anti-inflammatory, anti-neurodegenerative in addition to many other therapeutic classes (Donia et al., 2003; Rawat et al., 2006; Bao et al., 2007; Diers et al., 2008; Uemura, 2006). Some of the NPs isolated from marine invertebrates have been shown to be, or are expected to be of microbial origin (Tan, 2007). Marine microorganisms, whose immense genetic and biochemical diversity is only beginning to be appreciated, appear to serve as a rich reservoir of NCEs. The prokaryotic marine cyanobacteria are a potential source of structurally bioactive secondary metabolites. A number of cytotoxic compounds such as hectochlorin, lyngbyabellins, apratoxins, and aurilides have been identified as potential lead compounds for the development of anticancer agents (Jimeno et al., 2004; Braiia et al., 2006).

NATURAL PRODUCTS FROM MICROORGANISM The seemingly endless potential of microorganisms dawned upon mankind in 1928 with Alexander Fleming's sensational discovery of an antibacterial filtrate "penicillin" from a commonplace mould Penicillium notatum (Ligon, 2004). Re-isolation and clinical studies pioneered by Chain, Florey, and co-workers in the early 1940s, and the subsequent scale-up towards commercially viable manufacture of synthetic penicillins revolutionized the foundation of antimicrobial drug discovery (Bentley, 2005). The

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phenomenal success of penicillin encouraged drug companies and research groups to pool their resources together for a large scale culture of a diverse array of microorganism from which several newer antibiotics emerged (Knight et al., 2003). Early years of this large scale hunt for antibiotics yielded a number of successful antibiotics, some of which are widely prescribed to this day. Prominent among these include pathbreaking molecules in the history of modern medicine, such as streptomycin, chloramphenicol, tetracycline, cephalosporin, erythromycin, and vancomycin. Each one of these compounds, either in its original form or in its synthetic derivative, can be found on the shelves of a pharmacy to this day. The impact of these drugs has even served to change the very attitude towards infective diseases from one of horror and despair to that of a routine ailment of trivial consequence. More lives have been saved in history by antibiotics derived from nature, than through any other class of prescribed drugs. In other words, NP derived drug molecules have contributed the most towards increasing the life expectancy of man. Antibiotics of natural origin also remain some of the most often prescribed drugs in the world. Some of the earliest compounds discovered in the early 1970s using rational approaches based on mechanism of action, include the betalactamase inhibitor clavulanic acid from Streptomyces clavuligerus (Brown et al., 1976) and the HMG-CoA reductase inhibitor mevastatin (earlier code-named as ML-236B) from Penicillium citrinum (Endo, 1981). A mixture of clavulanic acid and amoxicillin (the combination is called Augmentin) is being used today as a front line antibiotic in several critical infections, while mevastatin and lovastatin, have been the leads in the discovery of a series of drugs in the treatment of dyslipidemia. Today, these groups of statins are among the top revenue earners for some of the richest drug companies in the world (Manzoni et al., 2002). The arrival of statins has also widened the scope of therapeutic possibilities in compounds isolated from microbes. Many biotech industries are now utilizing microorganisms as the raw material for the production of not only antibiotics, but also various enzymes, antibodies, vaccines and many genetic engineered hormones.

EXPLOITING NEW APPROACHES FOR NATURAL PRODUCT BY PHARMACEUTICAL COMPANIES Just as the arrival of aspirin revolutionised the preparation of medicines from an individual enterprise of laboratory dispensing into a scale of industrial manufacture, the invention of penicillin and other antibiotics ushered in a new era of large-scale manufacture through biotechnology. Although 60% of the new drugs owe its origin to NPs, this enormous pool of resources is being steadily overlooked by several large pharmaceutical innovators for over a decade (Baker et al., 2007). This strategy seems

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inappropriate because several synthetic analogues of the NPs have often proved themselves to be more efficacious and less toxic. For instance, chloroquine is less toxic than quinine, and homatropine is shorter-acting and therefore produces less cycloplegia than atropine. Lignocaine, the more therapeutically useful product, was produced by modifying the cocaine molecule. Semisynthetic penicillins like methicillin expanded the range of cure. Similarly, second and third generation synthetic cephalosporins have extended the antibacterial spectrum of the prototype. The advent of high throughput screening (HTS, see below) and the availability of combinatorial compound libraries have been attributed as major reasons for this change in attitude (Boldi, 2004). NP-based drug discovery has been finding it difficult in adapting itself to modern techniques such as HTS and rapid hit-identification process. This disadvantage is being felt most acutely by large pharmaceutical companies with enormous funds at their disposal. The long time-gap between drug discovery and its marketing tends to challenge the wisdom in this tendency to move away from natural products. However, the scenario will become clear only in the long term. Interestingly, smaller biotech companies are emerging with renewed strategies to focus on exploiting new approaches for NP-based drug discovery. These approaches include the application of genomic tools, identification of novel sources of organisms, newer technologies (listed in the Table 2) and improved processes of sample preparation for screening. In addition, the recent focus on the synthesis of diversity-oriented combinatorial libraries based on NP-like compounds is an attempt to enhance the productivity of synthetic chemical libraries (Marcaurelle et al., 2008). One of the breakthroughs in drug discovery was the use of mechanism-based screening for bioassay-guided fractionation (Phillipson et al., 2002). Efforts continue to improve screening formats, reagent production, robotics, and data management. Mechanism-based screening Table 2. Technologiestl'echniques applied in drug discovery from natural products Name of the technology! technique Chromatography: High performance liquid chromatography (HPLC) High performance thin layer chromatography (HPTLC) Gas chromatography (GC) Liquid chromatography (LC)

Applications Isolation of components from the natural sources. In vitro and in vivo drug metabolism and pharmacokinetic (DMPK) assays, HTS new molecule identification, Biomarker identification (Korfmacher, 2005; Castro-Perez, 2007)

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Table 2. Contd. Name of the technology! technique

Applications

Supercritical fluid chromatography (SFC)

For the isolation of chiral compounds from natural sources (Zhang et al., 2005)

Mass Spectroscopy (MS): GC-MS, HPLC-MS, HPLCMS-MS, LC-MS, Fluorescence activated cell sorting (FACS)-MS, Liquid chromatography photodiode array mass spectrometry (LC-PDA-MS)

Molecular identification, and the structural elucidation of natural products usually combined with GC or HPLC or LC (Ma et al., 2006; Koehn, 2008)

FTICR-MS: Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry

Biomarker discovery and analysis.Metabonomics, proteomics, natural product analysis and noncovalent complexes (including thermodynamics) (Feng et al., 2007; Zhang et al., 2005)

Nuclear magnetic resonance spectroscopy

Study on ligand binding to the protein, helps in the structure based drug design; Structural activity relationship (Robert, 2000; Coles et al., 2003; Pauli et al., 2005) Molecular isolation and identification usually combined with the HTS analysis (Koehn, 2008) For DMPK studies, HTS analysis of natural products (Corcoran et al., 2003) Studies of psychoactive agents in brain, Drug accumulation and metabolism in cancer, Quantitation and metabolism in the brain, liver and other organs (Paulus et al., 2007) To assess the biological function for unannotated proteins expressed by different genes with the protein structure solved (Reid et al., 2008; Powers et al., 2008)

(NMR)

Saturation Transfer Difference (STD)-NMR LC-NMR-MS, On-line solid-phase extraction (LC-SPE-NMR) Fluorine magnetic resonance (19F MR) Functional NMR (fNMR), Functional annotation screening technology by NMR (FAST-NMR) Positron emission tomography

Disease specific marker identification in nurological diseases (Wang et al., 2005; Beckmann et al., 2007; Hutchins et al., 2008)

High-throughput X-ray crystallography Wide-angle X-ray scattering

For protein structure studies, screening functional ligands, and ligand binding (Senior, 2005)

High-content highthroughput fluorescent microscopy

For the study of protein localization, dynamics and interactions with the natural products (Auer et al., 1998; Zemanova et al., 2003; Starkuviene et al., 2007)

has become the mainstay of HTS (Zheng et al., 2008). By utilizing new approaches, these companies are increasing the chemical diversity of their collections.

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SCREENING OF NATURAL PRODUCTS Traditionally, crude extracts have been used routinely for screening. Aqueous or alcoholic extracts are most frequently used for the initial round of biological and phytochemical screening. Recently developed bioactivity-guided fractionation processes can now separate crude fractions into mixtures containing only a few compounds. Column chromatography, highperformance liquid chromatography (HPLC), countercurrent chromatography (CCC), or centrifugal partition chromatography (CPC) are some of the techniques used for fractionation (Maugh, 1983). Fractionation will allow active compounds present in amounts too small to detect, whose activity is masked in a crude extract. However, by screening at higher concentrations, interfering compounds will also be more frequently identified. The paradox is that the total number of fractionated extracts screened is inversely proportional to the biodiversity. A decision must be made whether the loss of biological diversity is adequately compensated by fractionation. The answer to this question will help determine the optimal number of fractions that should be generated for each extract. The advent of sophisticated separation by analytical instruments attached to the HTS enables scientists to assemble large libraries of pure NPs, which can then be screened in a manner analogous to pure compound libraries (Liu et al., 2004). A recent review by McChesney et al. (2007) discusses latest methods and techniques involved in the natural product extraction, isolation and purification. The identification of the compounds that are responsible for the activity of an extract is called dereplication process (Konishi et al., 2007). This process prioritizes extracts for further study and can save considerable research time. The most generic procedure used today involves separation of an extract using reversed-phase HPLC and splitting of the eluent post column into a mass spectrometer (usually MSIMS to match fragmentation ions) and a fraction collector using micro-titer plates to test tubes, depending on the scale (Strege, 1998). The fractions are screened and the retention time, UV spectra, MS data, and activity of fractions are analyzed for common interfering compounds and known inhibitors using commercial and in-house databases (Schuffenhauer et al., 2005). Other methods for dereplication that have been used include separation by solid-phase extraction cartridges and CCC. Alternative methods for analysis include affinity capillary electrophoresis, LC-NMR, and online biochemical assay (Bobzin et al., 2000; Beger, 2006). An increase in the speed of bioassay-guided fractionation has been facilitated by a marked improvement in HPLC automation, MS, and column technology, as well as rapid turnaround of screening results provided by HTS (Mishra et al., 2008). The advent of new probe technology and higher magnetic fields has led to a significant shortening in acquisition time for

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NMR data, and the structure elucidation of NPs can be achieved routinely on amounts less than 1 mg (Liu et al., 2004; Xing et al., 2007). Automated structure-solving algorithms have made the job simpler but no technique can replace the structure elucidation skills of an experienced chemist (Ross et al., 2001). However, these programs add value to the tools that search for alternative structures that fit the same NMR data (Beger, 2006). IDGH THROUGH PUT SCREENING OF NATURAL PRODUCTS The discovery of truly novel NPs is a laborious and time consuming task when pursued through conventional programs such as extraction, fractionation, isolation and screening for biological activity. This has necessitated the adoption of high-throughput screening (HTS) by major players in the drug discovery team. HTS is defined as the automatic testing of potential drug candidates at a rate in excess of 10,000 compounds per week (Liu et al., 2004; Pereira et al., 2007; Koehn, 2008; Jenwitheesuk et al., 2008). HTS has gained widespread popularity over the last few years. The aim of high throughput drug discovery is to test large compound collections for potentially active compounds ('hits') in order to allow further development of compounds for pre-clinical testing ('leads'). In this approach, the molecular diversity and range of biological properties displayed by natural products is a challenge to combinatorial strategies for NPs synthesis and derivatisation (Posner, 2005). Many large companies study a hundred targets or more each year, and in order to validate these targets, lead compounds must be found. HTS is the process of assaying a large number of potential effectors of biological activity (lead) against the targets (a biological event) by miniaturized in vitro assays. Thus HTS technology has emerged over the last few years as an important tool for drug discovery and lead optimization in NP based drug discovery (Posner, 2005). HTS uses standard assay types known to most biological and biochemical scientists (e.g. ELISA, proliferation/cytotoxicity assays, reporter assays, and binding assays) (Inglese et al., 2007). The biochemical assay will be selected depending on the mechanism of action or target specificity of the compound or the mixture of compounds (commonly the fractionated extract). The primary assay is either cell free biochemical assays or cell based assays. Cell based assays allow the HTS to address a specific target (Castel et al., 2006). In order to process thousands of assays per day, HTS uses a combination of multiple well microplates and robotic processing. For a number of years, HTS assays have been run in the standard 96-well microplate (working volume of up to 250 ~L). The current goal of most companies is to move beyond this format to higher-density, lower-volume formats (e.g. 384 and 1536 well microplates). There are two primary advantages ofthese formats: increased throughput and lower volume, which

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translates into lower cost. At screening rates of 500,000 compounds/week, even a cost of $11 well becomes too big a burden on the company's weekly budget (Mishra et al., 2008). The reduction of cost, rather than increase in throughput, is the primary driving force within most HTS groups to move towards higher-density, lower-volume microplates. Fluorescence polarization (FP) detector technology is widely acceptable in the plate reader system (Burke et al., 2003; Rogers, 1997). For example screening assay for detection of apoptosis through activated caspase is assessed as liberation of free rhodamine following incubation of treated cells with rhodamine-labeled (quenched) substrate solution. Compounds showing a five fold or greater increase in fluorescence are considered as hits, being inducers of caspase activity. However, intrinsic fluorescence of natural products can interfere with such detection. Use of special reagents such as Europium-labeled reagents and time resolved fluorescence detection, avoids this problem (McMahon et al., 2000).

COMPOUND DEVELOPMENT: TOTAL SYNTHESIS, SEMISYNTHESIS OR COMBINATORIAL SYNTHESIS Perhaps the biggest obstacle to NP chemistry is the supply oflarge amounts of NPs. For instance, a steady supply of paclitaxel from nature would require cutting down a large number of whole yew trees followed by tedious extraction of the active molecule (McChesney et al., 2007). A sustainable source of the NP should be identified whenever total synthesis is not possible. However, a microbial source does not face such a problem on account of the brief life cycle of the organism and efficient culturing methods sustainable in lab conditions. This is precisely the reason why most companies prefer screening of microbial products. Rapid advances in microbial combinatorial biosynthesis have hastened the process in recent times (Dougherty et al., 2006; Adrio et al., 2006). There has also been progress with organisms usually considered to be problematic. For example, the steady supply of compounds from plants can be enhanced with minimal environmental damage through tissue culture and genomic methods (Wilkinson et al., 2005; Chaturvedi et al., 2007; GOmez-Galera et al., 2007). The study is now being extended to cover aquaculture of marine invertebrates and the identification, cloning, and expression of symbiotic microbial genes (Munro et al., 1999). The molecular complexity of NP derived NCEs can also impede the lead optimization process. A very important trend in recent years has been a significant effort devoted to the semisynthesis and synthesis of complex NPs (Ortholand et al., 2004; Shang et al., 2005; Paul et al., 2007; Bulger et al., 2008) (Listed Table 3). The success of this strategy is evident from history. Literature

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shows that a total of 322 NP-derived drugs have been launched from 1981 to 2006. More than 70% of these drugs (232 compounds) are manufactured by semisynthetic processes. Further, about 15% (47NCEs) have been produced by total synthesis (Newman et al., 2007; Butler, 2008). Paclitaxel, an anticancer drug manufactured by total synthesis, artemether, a semisynthetic drug from artemisinin, have emerged as synthetic breakthrough while vinblastine and vincristine (approved for cancer treatment in 1963 and 1965) templates are extremely difficult to synthesize economically. Table 3. Synthetic/semisynthetic analogues of natural products Name of drug

SourcelIndication

Acarbose

Hypoglycemic drug derivative of natural product from Aspergillus awamori

Alitretinoin

Anticancer; a first generation retinoid

Amrubicin

Derivative of doxorubicin from Streptomyces peucetius var caesius. Used in breast, lung, and gastric cancer

Anidulafungin

Antifungal agent; semi-synthetic lipopeptide from a fermentation product of Aspergillus nidulans.

Apomorphine hydrochloride

Synthetic analogue of morphine in Parkinson's disease

Arteether, artemether, artenusate

Semisynthetic derivative of artemesinin (from the plant Artemisia annual to treat malaria

Azithromycin

Macrolide antibiotic; Azane-substituent of erythromycin obtained from actinomycete Saccharopolyspora erythraea

Captopril, enalapril and cilazapril

Antihypertensive, developed from teprotide, isolated from Brazilian viper venom (Bothrops jararaca)

Carbepenams: Biapenem, Imipenam, Doripenem, Meropenam, Ertapenem Carumonam

Derived from a compound called thienamycin, produced by Streptomyces cattleya

Caspofungin acetate

Monobactam antibiotic, very resistant to ~-lactamase; derivative of aztreonam obtained from Chromobacterium violaceum Semisynthetic lipopeptide derived from pneumocandin BO, a fermentation product of Glarea lozoyensis; antifungal

Caspofungin acetate

Semisynthetic derivative of pneumocandin produced by fungi: Zalerion arboricola

Cefditoren pivoxil

An oral prodrug of cefditoren, a derivative of cephalosporin isolated from Cephalosporium species

Cladribine

Anticancer; related to nucleoside adenoside

Clari thromycin, Telithromycin, Dirithromycin

Macrolide antibiotic; Semi-synthetic erythromycin

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Table 3. Contd.

Name of drug Clindamycin Cytarabine ocfosfate

SourcelIndication Semisynthetic derivative of lincomycin, produced by the actinobacterium Streptomyces lincolnensis. Derivative of spongothymidine obtained from Caribbean sponge

Dicoumarol

Anticoagulant, derivative of plant coumarine isolated from fungal infected hay

Docetaxel

Anticancer; paclitaxel derivative

Ellipticine, Celiptium Epirubicin

Semisynthetic derivative from plant Ochrosia elliptica 4' -epimer of doxorubicin, an anticancer drug

Epirubicin HCI

Anticancer antibiotic related to doxorubicin

Etoposide phosphate

Anticancer; semisynthetic derivative from podophyllotoxin, found in the American Mayapple (Podophylum peltatuml

Everolimus

Orally active 40-0-(2-hydroxyethyl) derivative of rapamycin, originally produced from Streptomyces hygroscopicus.

Exemestane, Formestane

Anticancer; aromatase inhibitor structurally related to the natural substrate androstenedione

Exenatide

Antidiabetic: a synthetic analog of exenadin-4, which was originally isolated as a 39 amino acid peptide from the saliva of the Gila monster (Heloderma suspectuml

Fosfomycin trometamol

Antibacterial; analogue of fosfomycin obtained from Streptomyces fradiae.

Idarubicin hydrochloride (4demethoxydaunorubicinl

Anticancer antibiotic; derivative of daunourubicin

Irinotecan, topotecan

Camptothecin derivatives for cancer

Isepamicin

Aminoglycoside antibiotic; derivative of gentamicin B from Micromonospora, a genus of Gram-positive bacteria widely available in water and soil

Ivermectin

Anti-parasitic; derivative of avermectin obtained from microbe Streptomyces avermitilis

Micafungin sodium

Antifungal agent of the echinocandin type obtained from the culture broth of the fungus Coleophoma empetri ,and inhibits ~-(1, 3l-D-glucan synthase of fungi.

Micronomicin sulfate

Aminoglycoside antibiotic; 6'-N-methyl derivative of gentamicin C Antidiabetic; Acarbose related drug

Miglitol Miglustat

For type I Gaucher disease. An analogue of nojirimycin isolated from the broth filtrate of Streptomyces lavendulae

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Table 3. Contd. Name of drug

SourcelIndication

Miokamycin

Derivative of macrolide antibiotic miodecamycin Al ; 9,3'diacetylmiodecamycin; has wide antimicrobial spectrum

Mitobronitol (1, 6-dibromo1, 6-dideoxy-d-mannitoll

Alkylating anticancer; derivative of mannitol

Mycophenolate sodium

Mycophenolic acid, originally purified from Penicillium brevicompactum, used for prophylaxis in renal transplant

Nandrolone phenylpropionate

Anticancer; Analogue of naturally occurring nandrolone in human

Nitisinone

Derivative of leptospermone, an important new class of herbicides from the bottlebrush plant (Callistemon citrinus)

Orlistatin

Antiobesity; Semisynthetic product from lipstatin (a natural product)

Peplomycin

Semisynthetic analogue of bleomycin from bacterium Streptomyces verticillus

Pimecrolimus

A novel analog of ascomycin, isolated as a fermentation product of Streptomyces hygroscopicus var ascomyceticus, for allergic contact dermatitis and atopic dermatitis

Pirarubicin

Anticancer antibiotic similar to daunorubicin

Rifampicin

Semisynthetic compound derived from rifamycin which is obtained from Amycolatopsis rifamycinica

Rosuvastatin calcium

Hypolipidemic drug; derivative ofmevastatin isolated from Penicillium citrznum and P. brevicompactum

Telithromycin

Semi-synthetic derivative of the 14-membered macrolide, erythromycin-A, isolated from Saccharopolyspora erythraea

Testolactone

Anticancer; derivative of progesterone

Tigecycline

9-tert-butyl-glycylamido derivative of minocycline, which is a semi-synthetic product of chlortetracycline isolated from Streptomyces aureofaciens.

Tiotropium bromide

Synthetic derivative of atropine from Atropa belladonna (Solanaceae) to treat COPD

Triproamylin acetate

Antidiabetic; amylin analog

Valrubicin

Anticancer antibiotic similar to doxorubicin

Vapreotide acetate

Anticancer; Somatostatin analogue

Vindesine and vinorelbine

Anticancer; semisynthetic derivatives of vinblastine from herb Vinca rosea

Ziconotide

Non opioid analgesic; synthetic form of one of the orconotoxins, obtained from cone snail (Conus magus) venom

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Synthetic effort has been particularly critical for NP-derived anticancer drugs where total synthesis has been crucial in lead optimization and for obtaining enough material for clinical trials. Synthetic chemists are actively engaged in such ventures where there are 15 derivatives of paclitaxel, 15 of camptothecin, 4 of combretastatin, 4 of vinca alkaloids and 2 of podophyllotoxin in the clinical trial process (Saklani et al., 2008). With the advent of combinatorial chemistry, a large number of compounds can be produced from a single lead discovered from the NPs (Lee et al., 2001; Nielsen, 2002; Boldi, 2004; Ganesan, 2004; Tan, 2004). Combinatorial libraries based on natural product scaffolds, analogs and derivatives are being currently pursued. For example, combinatorial chemistry techniques applied to scaffolds oftubulin inhibitors, have yielded second-generation paclitaxel analogs as anticancer drug candidates (Ladame et al., 2008). Further, compounds active against vancomycin-resistant bacteria have emerged from combinatorial libraries of vancomycin- dimers (Griffin et al., 2003). The combinatorial libraries of anticancer molecules designed to target receptor tyrosine kinase, rest on the evidences obtained from a natural product genistein (Kissau, 2003). Thousands of camptothecin derivatives have been synthesized with the combinatorial approach to target topoisomerase I. Topotecan and irinotecan are derivatives obtained in this manner (Li et al., 2006). In spite of all the sophistication, combinatorial chemistry has failed to deliver the high numbers of drug leads as was predicted. This is mainly because of the complexity and diversity of the NP scaffold. Limited success has paved way for "diversity oriented combinatorial synthesis" which proposes to produce molecules with greater structural complexity including enhanced stereo-chemical variations (Rouhi, 2003; Liao, 2003; Tan, 2005; Marcaurelle et al., 2008), but these approaches may have limited prospects in future compared to the natural product based combinatorial library (Koppitz et al., 2006).

TRANSLATIONAL RESEARCH IN ASSESSING SAFETY AND EFFICACY OF BIOACTIVE NATURAL PRODUCTS: FROM BENCH TO BED SIDE In drug discovery and development programs, a vast majority of tested compounds do not progress from laboratories to patients' bedsides. Historical evidence indicates that only 14% of the tested products entering phase I trials eventually obtain clearance from the FDA and enter the market (DiMasi et al., 2003). In recent times, chances of success have reduced to just 8% (Graul, 2007). This high attrition rate is being attributed to the knowledge gaps between in vitro data, preclinical data and the final clinical outcome (Eisenstein et al., 2008). The basic knowledge about the investigational molecule or the natural product is not being translated

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appropriately to the clinical setting. The failures are not being analysed effectively together in a concerted manner by the drug discovery team and the clinical team. A two-way dialogue between scientists operating in preclinical drug discovery and clinical developers who understand the pathology and its alleviation, are required if novel leads and biologicals are to become useful drugs. Translational research, which focuses on such themes, has emerged as a new discipline in this context. The recent surge in translational research is expected to reduce the gap between the initial stages of drug discovery and subsequent clinical development (O'Connell et al., 2006). The American Physiological Society (APS) has defined translational research as 'the transfer of knowledge gained from basic research to new and improved methods of preventing, diagnosing or treating disease, as well as the transfer of clinical insights into hypotheses that can be tested and validated in the basic research laboratory (O'Connell et al., 2006). It is a kind of iterative learning experience from experimentation in basic and clinical fields. Thus in NP drug discovery, translational research interlinks preclinical and clinical research, where experiments progress steadily from lead generation towards proof of concept (early-stage clinical studies) (Sultana et al., 2007). Further, translational research assumes a broad perspective, embracing diverse areas such as clinical demands, public health policies and economic compulsions (Trusheim et al., 2007). The preclinical studies carried out in animals can predict efficacy (confidence in rationale) and safety (confidence in safety) of compounds or the natural product extracts in early development. The animal models and biomarkers (see below) can only give a preliminary idea of clinical correlation. Further, this may be poorly correlated to clinical outcomes in man. The pharmacokinetics and pharmacodynamics (PK-PD) of the compound in humans, which becomes clear only after the first dose in healthy volunteers, will help in determining the dose and route of administration. Research in normal human volunteers and patients enables a clearer understanding of the biology and pathophysiology, and can therefore trigger innovations in in vitro and animal experiments that better mimic the clinical condition. This strategy is therefore likely to enhance confidence in the appropriateness of a given target and help in dose selection for subsequent trials (Sartor, 2003). Biomarkers are quantitative measures of biological effects that provide informative links between mechanism of action and clinical effectiveness (Wehling, 2006). Assessment of biomarkers can reduce the uncertainty around key risks in the drug development cycle (pharmacology, PK-PD, toxicity, safety etc.). Further, biomarkers reflect biological effects induced by disease! test-compound and are the main tools to predict and describe its efficacy and safety. Specific markers like serum enzymes (ALT,

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AST, Catalase, Alkaline phosphatase etc) and more general markers of disease-severity like C-reactive protein in inflammatory diseases are well known biomarkers. Histological parameters, imaging of tumors and the morphology of atherosclerosis, liver fat (safety biomarker), positron emission tomography (brain disorders) scans etc. are some disease-specific biomarkers (Colburn, 2003). In preclinical studies, death could be a biomarker in models of myocardial infarction, cancer, acute toxicity etc., being correlatable measures of efficacy. These biomarkers are used to achieve predictive power and this can vary from almost useless (e.g. serial brain slices etc., which can never be a human biomarker) to surrogate (e.g. LDL cholesterol, one of the few surrogates accepted by FDA), with most cases falling somewhere in between. Translational research initially was said to be bidirectional but it has got multidirectional approaches (Sonntag, 2005). It has an important role in identifYing new indications for established therapies - a process termed by some authors as 'indications discovery'. New drug target hypotheses can be generated through preclinical experiments on novel target organs. It can also result from clinical observations such as side effects or additional pharmacological effects beyond those expected from efficacy in the main indication (O'Connell et al., 2006). Sildenafil, a selective Phosphodiesterase5 inhibitor, is an example of a drug initially assessed for angina pectoris but was subsequently licensed for a completely different pathology; male erectile dysfunction and pulmonary artery hypertension (Ghofrani et al., 2006).

NATURAL PRODUCTS FOR TYPE 2 DIABETES: A CASE STUDY Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The prevalence of diabetes for all age-groups worldwide was estimated to be 2.8% in 2000 and 4.4% in 2030. The total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030 (Wild et al., 2004). Type 2 diabetes (T2D) is prevalent in 90% of the patients with diabetes. By 2025, more than three-quarters of all persons with diabetes will reside in developing countries. India and China are leading this surge in diabetes, and sub-Saharan Mrica is currently at a lower prevalence rate (Dagogo-Jack, 2006). Poorly managed diabetes leads to several complications (e.g. end-stage renal failure, blindness, amputation and heart disease) that many developing countries are ill equipped to tackle (Home, 2003). Diabetes is a heterogeneous metabolic disorder characterized by a decline in insulin action (insulin resistance), followed by pancreatic beta cell dysfunction (Chiasson et al., 2004). Insulin resistance will be associated with common conditions such as metabolic syndrome, dislipidaemia, obesity,

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endocrine disorders and hypertension. Abnormal obesity, increased lipid profiles, atherosclerosis and hypertension are symptoms of the metabolic syndrome (Wild et al., 2007). Prescription drugs available today reduce blood glucose level either by increasing the tissue uptake or by increasing the insulin secretion. There is no successful drug to treat the metabolic complexes associated with diabetes (Matfin, 2008). A wide range of natural products are prescribed to the diabetic patient as nutraceuticals or dietary supplements for the intervention of disease, treatment and/or prevention (for more details http://www.naturaldatabase.com). Compounds of chromium, selenium, vanadium, herbal preparations of garlic, ginseng, a-lipoic acid and various mixed-herbal products are among the dietary supplements that are currently being used by many diabetics (Table 4). A recent review by Hays et al. (2008) discusses the rationale behind prescribing these natural products. Despite compelling evidence supporting an important role for dietary chromium in the restoration of normal glucose tolerance among chromium- deficient individuals, the ability of chromium to improve insulin action remains controversial (Balk et al., 2007). While a number of animal models have generally demonstrated anti-diabetic properties for ginseng, relatively few clinical studies in humans have been reported. Taken together, research data suggest that ginseng is likely to show a beneficial effect on certain metabolic parameters in T2D (Xie et al., 2005). However, it is difficult to make standardized clinical recommendations on account of the large reported variability in metabolic responses to different ginseng species, different batches of the same species, and different parts of the ginseng plant. On the whole, standardization of natural products remains a major bottleneck in the search for clinical evidence.

Table 4. Commonly used conventional and natural products for diabetes Prescription drugs

Natural products

Hypoglycemic agents Chlorpropamid Glimepiride Glipizide Glyburide Nateglinide Repaglinide Tolazamide Tolbutamide

Banaba (Lagerstroemia speciosal Bitter melon (Momordica charantial Fenugreek (TrigoneUa foenum-graecuml Gymnema (Gymnema sylvestre)

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Table 4. Contd. Prescription drugs

Natural Products

Insulin sensitizers Metformin Pioglitazone Rosigli tazone

American ginseng (Panax quinquefolius) Banaba (Lagerstroemia speciosa) Cassia cinnamon (Cinnamomum aromaticum) Chromium Ginseng (Panax ginseng) Prickly pear cactus (Opuntia ficus-indica) Soya (Glycine max) Vanadium

Carbohydrate absorption inhibitors Acarbose Miglitol

Miscellaneous Exenatide Pramlintide Sitaglitpin

Blond psyllium (Plantago ovata) Fenugreek (Trigonella foenum-graecum) Glucomannan (Amorphophallus konjac) Guar gum (Cyamopsis tetragonoloba) Oat bran (Avena sativa) Prickly pear cactus (Opuntia ficus-indica) Soy (Glycine max) a-lipoic acid Selenium Stevia (Stevia rebaudiana)

Numerous cell culture and in vivo animal studies have demonstrated that treatment with the water soluble antioxidant a-lipoic acid is associated with improved skeletal muscle glucose transport activity, reduced wholebody insulin resistance, and reduced oxidative stress. Although relatively few clinical trials have been reported, the combination of compelling in vitro and in vivo animal data in the presence of limited clinical data indicate the need for further research in human subjects while only supporting a general recommendation for a-lipoic acid supplementation (Bilska et al., 2005). Epidemiological studies on various populations living on a diet consisting of a high proportion of fatty fish (a rich source of omega-3 fatty acids) demonstrate a lower incidence of T2D compared to matched population groups. Positive changes in lipid profile and blood pressure demonstrated by epidemiological and other studies suggest that omega-3 fatty acid supplementation may reduce cardiovascular disease risk in T2D patients, even if no direct improvement in insulin or glucose homeostasis has been conclusively demonstrated (Lombardo et al., 2006). Many Indian medicinal plants have shown marked efficacy in the treatment of diabetes (Saxena et al., 2004; Modak et al., 2007). A recent review by MukheIjee et al. (2006) has discussed the Indian medicinal plants

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with antidiabetic activity. However, detailed clinical investigations on the simultaneous use of prescription drugs along with natural products and dietary supplements are required for ensuring clinical safety and efficacy. A thorough preclinical and clinical investigation is necessary for establishing evidence. Diabetes presents a singularly complex condition where multiple targets operate in conjunction with the very complex physiology of nutrition and energy homeostasis. The number of druggable targets in diabetes is steadily increasing, with more and more successful drug classes entering the market. For instance, the DPP-IV (Dipeptidyl peptidase IV) inhibitor sitagliptin has recently been launched. While synthetic compounds address these targets individually, natural products probably tackle them in a collective and perhaps more holistic fashion. Restoring the equilibrium in the disturbed scenario of metabolic syndrome is probably linked to the ability to manage multiple targets taken together. While the effect on each individual target is sub-clinical, the collective pharmacodynamic picture is likely to playa role in restoring the complex metabolic equilibrium. For instance, we have demonstrated in our labs that extracts of Dodonaea I ~ t ~

f

SpeCific gene expression in

adlpocytes

.t- FA uptake '" Lipolysis

~

c==z:> ~ FFA

~oo~ t

II

- - _.......-

Insulin

'---V

sensitizer

.

factors

~ I

Expressionl action

.

"

~+

of insulin resistance factors

e.g. Reslstin! TNF

e ROS viz., ·OH, O2• •, Lpx Glucose Uptake

Fig 1. Schematic representation of the possible mechanisms by which a typical NP affords antidiabetic activity (PPAR y. Peroxisome Proliferator-Activated Receptor-Gamma; FA: Fatty Acids; FFA: Free Fatty Acids; TNF: Tumour Necrosis Factor; PTP-1B: Protein Tyrosine Phosphatase 1B; GLP-l: Glucagonlike peptide-1B; GIP: Gastric inhibitory polypeptide); DPP IV: Dipeptidyl peptidase IV; ROS: Reactive Oxygen Species; ·OH: Hydroxyl Radical; O 2 • -: Superoxide Radical; Lpx: Lipoxide Radicals)

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viscosa and Ficus racemosa stem bark show antidiabetic activity through multimodal mechanisms in animal models. The possible mechanisms suggested are increase in binding of PPAR-y (Peroxisome ProliferatorActivated Receptor-gamma), inhibition of PTP-1B (Protein tyrosine phosphatase 1B), DPP-IV (Dipeptidyl peptidase IV), IX-amylase, IX-glucosidase, tissue glucose uptake and reduction in the oxidative stress (Unpublished data, See Fig 1). The pharmacodynamic complexity is fitly matched by the diversity of targets addressing euglycemia in the body. While restoring or correcting these anomalies individually, one at a time, is the objective of conventional antidiabetic therapy, herbal products appear to tackle many of these targets in a concerted manner, providing a more sustainable method with perhaps a lower incidence of adverse events (Veerapur, 2007). REVERSE PHARMACOLOGY: AN EFFECTIVE STRATEGY The beaten track of drug discovery rests on the paradigm that the course of innovation begins in the lab and ends in the clinic. Reverse pharmacology is a complete turnaround in strategy, where clinical evidence works at the root of drug discovery (Patwardhan et at., 2004). It is essentially a clinic to lab strategy, working backwards, where existing claims by traditional systems of medicine, such as Ayurveda, give clues to possible clinical value of existing formulations in traditional medicine. In such situations, clinical safety and proof of concept are the premises upon which the program is built, thus saving millions of dollars that are customarily spent on ensuring clinical safety and meeting regulatory rigours. Consequently, efficacy becomes a matter of validation, with the help of scientific evidence gathered from laboratory research. It is therefore a confluence of traditional wisdom, modern medicine and science. If exploited intelligently, it becomes a viable mode of drug discovery where holistic systems and traditional wisdom are integrated with laboratory evidence and modern science. The major advantage of reverse pharmacology is that it helps in reducing three major bottlenecks that plague the drug discovery programmes viz., time, money and toxicity. These bottlenecks are major hurdles in drug discovery impeding not only the pace of its progress but also increasing the costs. Increase in regulatory rigour has become one of the major reasons for the ever diminishing entry of NCEs. In this context, reverse pharmacology appears to be a viable alternate strategy. Several industries have realized the need for reinventing the drug discovery strategies for innovating with value added products.

Ayurveda and such other traditional systems of alternate medicine have a rich knowledge base with a vast materia medica consisting of thousands of plants, minerals, animal products used singly or in combinations, with various processes for the formulation of medicines. Most

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of these formulations and therapeutic strategies have survived on the basis of evidence that have sustained centuries of clinical experience. However, lack of standardization and validation continues to undermine the pharmacopoeias of alternate medicine. It is in this context that modern medicine and science can act as a complementary support for establishing proof of concept and validation of claims.

CONCLUSIONS The last three decades have witnessed an unprecedented worldwide growth in the interest in NP- based medicines, both in developed and developing countries. Historical evidence shows that NPs offer a resource-pool for lead identification in drug discovery. NPs have a reasonable likelihood of biological activity, and isolation of a novel molecule is easier than de novo synthesis, especially when the molecular structure of the compound is very complex. NP-derived molecules are amenable to semisynthetic modifications and derivatisation. Such compounds are also significantly more complex and novel than most synthetic compounds, in addition to being chirally pure. Chiral specificity is very much in tune with the physiology of the body, giving the drug candidates a far greater level of specificity than nonchiral molecules. On account of such advantages, pharmaceutical companies are opting for NP based drug-leads. At present there is limited data on safety, efficacy, and clinical trials to prescribe NPs in therapy. However, the NP market has been continually growing at an increased rate during the last 30 years, mainly as nutraceutical, dietary supplements, cosmoceuticals and a few of them as chemopreventives. NP based therapy should emphasise on well-controlled and randomized clinical trials to prove safety and efficacy. International regulatory guidelines have to be followed in order to harmonise the use ofNPs. In some countries, the newer strategies and guidelines have been tried for the NPs (Briggs, 2002; Barnes, 2003; Vaidya et at., 2007; Calapai, 2008). Reverse pharmacology, which is driven by clinical evidence drawn from ancient wisdom, rather than preclinical data, also offers a viable time and cost saving strategy. Emphasis should also be given to the production and genetic improvement (whenever possible) of medicinal plants and other NPs. Recombinant DNA technology offers opportunities to synthesise the cryptic molecules of nature in a simple environmental-friendly way. Further, the emphasis must shift towards discovering leads from unexplored realms of biodiversity (Harvey, 2000; Harvey, 2007). Alternative sources for natural products as also the techniques to save the extinction of biodiversity should be given due importance for sustaining the program (Kennedy, 2008). Finally, but not any less important, is the need for a more detailed legislation on intellectual property rights related to the marketing of NPs. All these strategies are urgently needed to avoid both business conflicts and territorial conflicts on biodiversity.

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2 The Modern Application of Traditional Chinese Medicine: Indigo N aturalis as an Alternative Therapy in Psoriasis vulgaris YIN-Ku LIN 1 ,2 AND JONG-HWEI Su PANG2 ,*

ABSTRACT

Psoriasis is a distressing, recurrent disease that significantly impairs the quality of life and has no permanent cure. Traditional Chinese medicine is an alternative method of therapy that can be used in the psoriasis treatment. It has been reported in Chinese literature that indigo naturalis exhibited potential anti-psoriatic effects in systemic therapy. However, its pharmacology was unclear and its gastrointestinal side effects limited its use in clinical practice. This article reviews and summarizes our experience of psoriasis therapy using Chinese herb prescription and the investigation of the anti-psoriatic mechanism of indigo naturalis. Indigo naturalis formulated in ointment has efficacy in treating psoriasis and can avoid adverse systemic effects. The possible mechanism of indigo naturalis action in treating psoriasis includes inhibiting hyperproliferation of epidermal keratinocytes and lymphocytes infiltration, as well as restoring epidermal differentiation and tight junctions in psoriatic skin. We believe that indigo naturalis will become a promising natural product that exerts great therapeutic potential as an alternative therapy in psonas£s. Key words: Traditional Chinese medicine, psoriasis, indigo naturalis, indirubin, keratinocyte, differentiation 1. Department of Traditional Chinese Medicine, Center for Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung, 204, Taiwan. 2. Graduate Institute of Clinical Medical Sciences, Chang Gung University, Tao-Yuan, 333, Taiwan. * Corresponding author: E-mail: [email protected]

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INTRODUCTION Psoriasis is a chronic inflammatory disease of the skin characterized by generalized erythematous papules or well-demarcated erythematous plaques topped by silvery scales which vary in severity and clinical manifestation. The most common form, plaque psoriasis, affects 2-3% of the Caucasian population (Lebwohl, 2003) but only 0.3% of the Mongoloid population (Yip, 1984). Many factors such as genetic features, immunological disorders, biochemical changes, environmental effects, trauma, stress, endocrine features, infection, drugs and alcohol play important roles in the pathogenesis of psoriasis (Ortonne, 1996). Conventional treatment for psoriasis has several disadvantages, including disease rebound or worsening after treatment is stopped (e.g. steroid) and risk for organ toxicities, such as nephrotoxicity and hepatotoxicity (e.g. cyclosporine, methotrexate). The fear of adverse events in long-term use may lead to poor compliance and results in following flares. Therefore, it is important to explore an alternatively new intervention which is relatively safe and can prolong the remission duration of psoriasis or prevent progression. The earliest description of psoriasis in ancient Chinese medical literature may be found in the 5th century AD. Traditionally, Chinese herbal preparation has been extensively used to treat psoriasis both topically and orally for centuries, and produced promising clinical results (Bedi & Shenefelt, 2002). Most of the key actions of Chinese herbs commonly used in treating psoriasis reflect anti-inflammatory properties, modulation of cytokine production or inhibition of angiogenesis (Tse, 2003). All these actions are potentially relevant in reducing the severity of psoriasis. Nowadays, traditional Chinese medicine (TCM) is still one of the most frequently chosen alternative therapies that can be used in the treatment of psoriasis in China and Taiwan. However, its lack of randomized blinded control trials and its underlying mechanisms of action have not been systemically investigated (Koo & Desai, 2003). The theoretical system and the terminology ofTCM are quite different from those of Western medicine. The doctors examine their patients with four traditional diagnostic methods (inspection, auscultation, interrogation, and pulse-taking), determine the principle of treatment according to the differentiation of symptoms and signs, and prescribe a formula according to the principle of treatment. In general, standard TCM practice emphasizes the importance of using many herbs that are combined in different formulations for each individual patient, sometimes mixing more than 10 different herbs. The mixture of herbs prescribed varies individually depending on the subtype of psoriasis (''blood-heat'' type, "blood deficiency dryness" type, and ''blood stasis" type), which is determined in traditional Chinese by many findings, including the lesions of psoriasis, the pulse, and the condition of the tongue (Tse, 2003). This is vastly different from Western medicine, which uses therapeutic approaches that are standardized and mono-therapy, as well as emphasizing

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"average" efficacy in large, double-blinded, placebo-controlled studies. Due to the overwhelming variability in how patients are treated, both between TCM practitioners and within the practice of a single TCM practitioner, it is difficult to conduct controlled trials to study traditional approach of individual polypharmacy. Indigo naturalis is one of the Chinese herbal remedies that have been reported to exhibit potential anti-psoriatic efficacy (Yuan et a!., 1982). However, the poor solubility and low absorption of indigo naturalis, and the adverse effects associated with irritation of the gastrointestinal tract and liver function in long-term systemic use have been occasionally reported (Verucchi et al., 2002). In order to avoid the adverse systemic effects, but retain the demonstrated efficacy of indigo naturalis as an anti-psoriatic medicine, we initiated an alternative approach in 2003 by applying the drug topically on skin lesions. Within the last five years, we have been using indigo naturalis formulated in ointment topically to treat thousands of patients with psoriasis. We also used indigo naturalis as a mono-therapy in treating psoriasis and conducted randomized, observer-blinded, vehicle controlled, intra-patient comparison trials to evaluate the clinical efficacy and safety in patients with psoriasis treated with indigo naturalis. This unique mono-therapy, rather than individual polypharmacy that TCM used to emphasize, hence provided a compelling evidence of indigo naturalis for treating psoriasis. This review aims to provide our clinical experience for the efficacy, safety of indigo naturalis in topical treatment of psoriasis, and our studies on the pharmacology of indigo naturalis and its active components.

INDIGO NATURALlS'S PROFILE Indigo naturalis (Qing Dai) is a dark blue powder originating from the branches and leaves of various indigo-producing plants, such as Strobilanthes fiaccidifolius, Polygonum tinctorium, Baphicacanthus cusia Bremek, and Isatis indigotica Fort. The extraction process is based on the natural fermentation method in Taiwan. In brief, the freshly cut plants are soaked in water until they decompose and the contents of indigo naturalis are ready to be extracted. An adequate amount of limestone powder is then added and oxygen is forced into the liquid usually by stirring the soaking solution thoroughly until it turns from blackish green to deep red, extracted products contained in the foaming surface of the mixture are collected and air-dried for later use. Indigo naturalis was used as textile dyes, paint pigment and medicine in ancient China. Indigo naturalis and indigo concoctions are recorded as being applied externally or taken internally before the first century AD in China and India. Until the mid-twentieth century, when antibiotics and steroids came into the world, indigo naturalis was one of the most important

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and efficacious treatments to heal ailments of various infections and inflammatory diseases in China and Taiwan. There are many people today who still have faith in the powers of indigo naturalis to cure various difficult illnesses, such as cancer. In TCM, indigo naturalis has been described as possessing antiinflammatory, anti-microbial, analgesic and sedative properties, i.e. "clearing away heat, detoxicating, removing blood-heat and ecchymoses, purging liver-fire and arresting convulsion". Indigo naturalis has been used as a remedy for high fever in infectious sickness, infantile convulsions, epilepsy, aphtha, mumps, pharyngitis, tonsillitis, gingivitis, periodontitis, and various dermatoses, including eczema, eruption, erysipelas, carbuncles, furuncles and insect or snake bites. Modern research, particularly in China, is revealing that indigo naturalis, with effects of antivirus, and anti-bacteria, has also been used as an important source of anti-cancer drugs in China (Han, 1994). A well-studied example is Danggui Longhui Wan, a mixture of 11 herbal medicines traditionally utilized against certain types of leukemia. Only one of these ingredients, indigo naturalis, was found to carry the antileukemic activity (Xiao et al., 2002). Although indigo naturalis is mostly constituted of indigo blue, a minor constituent, indirubin, was identified as the active component (Hoessel et al., 1999).

TOPICALLY INDIGO NATURALIS IN PATIENTS WITH PSORIASIS Clinical Efficacy Since 2003, we began to use Chinese herbs topically to treat patients with psoriasis. Initially, we used a combination of three herbs, indigo naturalis, Scutellaria baicalensis-Georgi, and Cortes phellodendri, which was recommended for psoriatic treatment in TCM. The topical ointment combined mixed herbs and vaseline, yellow wax as well as olive oil. We found that many patients with psoriasis, after multiple treatment failures with conventional anti-psoriasis medications, showed clinical improvement after topical treatment with indigo naturalis composite ointment. In addition, remission time was found to last longer than their previous therapies. This novel finding was reported in the journal Pediatric Dermatology (Lin et al., 2006). An 8-year-old boy with widespread psoriasis showed remarkable improvement with 8 weeks of topical treatment with this ointment. The psoriatic lesions on his body disappeared completely and the total body surface area involvement (BSA) decreased from almost 80% to 0%. Remission has lasted for over 2 years. In past 6 years observation, only mild disease recurred with no more than 1% ofBSA was noticed in this patient which was immediately improved after re-treatment with indigo naturalis composite ointment. Because this formula consists of three herbs, it is difficult to conduct controlled trials and investigations of pharmacological mechanism. We reviewed the published literature, and

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found indigo naturalis and its active ingredients, indirubin, have been orally used to treat psoriasis with considerable effectiveness (Koo & Arain, 1998). Therefore, we initiated the indigo naturalis mono-therapy for psoriasis and we reported that two adult patients with severe, recalcitrant psoriasis were successfully treated with topical indigo naturalis in the journal Clinical and Experimental Dermatology (Lin et al., 2006). Two patients were male aged 45 and 36. One had an 8-year history of psoriasis and was presented with 40% BSA and a Psoriasis Area and Severity Index (PAS!) score of 28.8. The other patient had suffered psoriasis for 22 years. He was presented with more than 80% BSA and a PASI score in excess of 42. The treatment by indigo naturalis ointment resulted in an approximately 95% reduction in PASI scores and BSA in these two patients within 3 months (Fig 1).

Fig 1. Clinical improvement after the treatment with indigo naturalis. A 40-year-old man with a 22-year history of plaques-type psoriasis . (A) Severe plaques are salmon pink in color, covered by silvery scales on right arm at the initial visit; (B) the same arm after 12 weeks of treatment with indigo naturalis ointment

In order to provide evidence-based data for this alternative therapy in psoriasis, we designed a randomized, vehicle-controlled clinical trial to evaluate the efficacy and safety of topically applied indigo naturalis in treating plaque-type psoriasis. An 8-week pilot clinical trial was performed during April-June 2004 in our hospital. Fourteen patients (10 men and 4 women) with chronic plaque psoriasis were enrolled. The mean patient age was 35.8 years (range, 21-54 years) and the median disease duration

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was 10.8 years (range, 2-30 years). The median pretreatment PASI was 11.7, with a median BSA of 13.6%. The patients were topically treated with either indigo naturalis ointment or vehicle ointment on contralateral skin lesions daily for 8 weeks. Efficacy was evaluated on the basis ofthe clinical scores, including induration, scaling, erythema and clearing percentage. Ten patients completed the trial and the results demonstrated a significant reduction in clinical scores achieved by topically applied indigo naturalis ointment. This interesting result has been published in the journal Dermatology (Lin et al. , 2007). To further confirm the efficacy of indigo naturalis for psoriasis treatment, another randomized, observer-blinded, vehicle controlled, intra-patient comparative trial was conducted. Fortytwo (32 men, 10 women) outpatients with chronic plaque psoriasis were enrolled. The mean patient age was 34.6 years (range, 18-58 years) and the median disease duration was 10 years (range, 2-41 years). The median pretreatment PASI was 14.7, with a median BSA of 18%. Target lesions were located on the upper extremities (17 participants), lower extremities (21 participants), and trunk (4 participants). The patients applied either indigo naturalis ointment or vehicle ointment topically to each of two bilaterally symmetrical psoriatic plaque lesions for 12 weeks. There was no significant difference in score of area, redness, thickness, and scaling between lesions which were assigned for treatment by indigo naturalis and vehicle ointments in baseline. The outcomes were assessed with the sum of erythema, scaling, and in duration scores and the clearing percentage of the target plaque lesion by two blinded professional observers. A significant reduction in the sum of scaling, erythema and in duration scores (p
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other currently used topical medicine (e.g. corticosteroid) to further evaluate the response duration and the disease recurrence. Histological Response to Indigo Naturalis Treatment in Psoriatic Lesions Based on our histological examination of skin biopsies, we found a marked decrease in epidermal thickness and elongated rete ridges for the lesions treated with indigo naturalis compared with the untreated lesions (Fig 2). We suggested that the anti-psoriatic effect of indigo naturalis might be mediated by controlling hyperproliferation and/or abnormal differentiation of epidermal keratinocytes, and/or exhibiting immunomodulatory effects in psoriatic lesion. We analyzed and compared the expression of different marker proteins with respect to proliferation (Ki-67, PCNA), differentiation (involucrin, filaggrin), and inflammation (CD3) in psoriatic lesions treated with indigo naturalis ointment and vehicle ointment. Results from immunohistochemical analysis showed a marked decrease in the number of cells with positively Ki-67 -stained nuclei in the indigo naturalis ointment-treated lesions compared to that in vehicle ointment-treated lesions (Lin et al., 2007). Similar findings were also demontrated for PCNA marker (Lin et al., 2007). The increased and normalized expression of filaggrin was observed in the stratum coreum and granular layer of the skin lesions treated with the indigo naturalis ointment (Lin et al., 2007). Involucrin also expressed normally in the granular and upper spinous layers after indigo naturalis therapy (Lin et al., 2009). In contrast to the original psoriatic lesions, the number of positively CD-3-stained T lymphocytes was demonstrated to be significantly reduced in the epidermis and papillary dermis of indigo naturalis ointment-treated lesions (Lin et al., 2007). In addition, based on our histological examination of skin biopsies, we found the presence of wide intercellular clefts only in the epidermis of lesions before treatment and control lesions treated with the vehicle ointment, but not in lesions treated with indigo naturalis ointment. In the treated psoriatic lesions that showed good clinical improvement, the keratinocytes in the granular layers were found to adhere to each other tightly. This finding implied that the impaired epidermal tight junction of psoriatic lesions may recover as a result of indigo naturalis treatment. We analyzed the expression of different marker proteins with respect to tight junction function (such as daudin-I) in psoriatic lesions, and the results showed a significant increase and recovery after treatment with topical indigo naturalis ointment compared with vehicle ointment (manuscript submitted).

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Fig 2. Effect of topically applied indigo naturalis ointment on the histological change of psoriatic lesion. Skin biopsies taken at baseline and after 8 weeks of treatment were processed for histological examination by HE stain method. (A) Untreated psoriatic lesion. (B) Indigo naturalis-treated lesion. Original magnification: xlOO

In short, our pathological findings suggested that indigo naturalis has great potential values in the treatment of psoriasis by inhibiting hyperproliferation of epidermal keratinocytes and lymphocytes infiltration, as well as restoring epidermal differentiation and tight junctions in psoriatic skin. Whether these findings are simply a result of skin recovered from psoriasis after indigo naturalis treatment or indigo naturalis might directly modulate these changes in skin is waited to be clarified by investigating the pharmacology of indigo naturalis in much more details.

Adverse Events Indigo naturalis, as a crude extract, appears to be dark blue in color and has a slightly unpleasant odor. The indigo naturalis ointment might slightly

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stain the skin and clothing which can be cleaned thoroughly by common detergents. Repeated application has no significant effect on the skin color and does not change the skin appearance. However, either the color or smell of indigo naturalis may affect the compliance offew patients, therefore benefit comes along with longer treatment can not possibly attain for them. Among 10 patients who completed an 8-week pilot clinical trial, 2 patients reported a short period (3-5 days) of itching at the beginning of treatment. In the following treatment period, no further adverse events such as scaling, erythema, tenderness, vesicles or other serious problems were found (Lin et al., 2007). Another 12 weeks clinical trial, 4 of 34 patients who completed the treatment reported itching after applying the indigo naturalis ointment to their psoriatic plaque, but the itching was only present for a couple of days at the start of treatment, and no tenderness, vesicles or other serious problems were found in the subsequent treatment period (Lin et al., 2008). In these two study groups, there were no abnormalities seen in the liver function tests, renal function tests, and complete blood counts after trial. Similar results were obtained in our long-term follow-up of these patients for the past 5 years. In our past 5-years experience in clinic, there were no serious events such as hepatic toxicity, renal damage and gastrointestinal problems found in these studies. Hypersensitivity, however, may be the major adverse effect as we have noticed in clinic (less than 15%). Some patients developed erythrodema and itching after using topical indigo naturalis treatment for psoriasis and most of them could recover by discontinuing the indigo naturalis application. Combining the treatment with topical steroids intermittently the development of allergy contact dermatitis may also be avoided or diminished.

PHARMACOLOGICAL PROPERTIES OF INDIGO NATURALIS Although the effectiveness of indigo naturalis in the treatment of psoriasis is well established, the mechanism of its action is poorly understood. We have utilized cultured normal human epidermal keratinocytes (NHEKs) as a psoriasis-relevant experimental model to investigate the potential effects of indigo naturalis on the gene expression of keratinocytes. In our laboratory data, we found that indigo naturalis could suppress proliferation and promote differentiation of epidermal keratinocytes which were correlated with the down-rgulation of PCNA gene expression and the upregulation of involucrin gene expression of NHEKs (Lin et al., 2009). We have also made another novel finding that indigo naturalis can improve skin barrier protection, at least in part, by up-regulating claudin-1 gene expression and therefore the tight junction function (manuscript submitted). The main components of indigo naturalis used in our clinic are indigo blue, indirubin and tryptanthrin as identified in chromatographic fingerprints. Among them, indigo blue in the most abundant one, however,

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the activity in psoriasis treatment has not been reported yet (Lin, 1993). Our preliminary data suggest that indigo blue may exert an antiangiogenic effect which may also partially explain the efficacy of indigo naturalis. Indigo blue extracted from some of the indigo-producing plants was reported to possess a hepatoprotective effect (Sreepriya et al., 2001). Indirubin and its analogues have been used to treat psoriasis orally in China (Lin, 1993). In addition, clinical trials showed that indirubin has a definite efficiency against chronic myelogenous leukemia (Han et al., 1988; Xiao et al., 2002). Indirubin and its analogues selectively inhibit CDKs and other kinases and block cell proliferation in the late G 1 and G2/M phase of the cell cycle (Marko et al., 2001). The promotion of neutrophilic differentiation of human myelocytic leukemia HL-60 cells by indirubin has also been reported (Suzuki et al., 2005). Furthermore, recent studies have revealed that indirubin simultaneously targets several signaling/molecules involved in the pathological process of cancer. The targets include those involved with cell cycle regulation (Hoessel et al., 1999), growth factor receptor signaling (Stat3) (Nam et al., 2005) and AhR signaling (Knockaert et al., 2004). However, mechanism underlying the efficacy of indirubin in psoriasis is still unclear, and the pharmacology of indirubin in keratinocytes also is unknown. Tryptanthrin, another bioactive ingredient from indigo-producing plants, has been reported to exhibit anti-tumor, anti-inflammatory and antiallergic activities (Lin, 1993). Whether tryptanthrin play any role in the anti-psoriatic effect of indigo naturalis is not elucidated and we will further investigate its pharmacology. SUMMARY

Although the western medicines currently used for psoriasis treatments can produce clinical improvement at controlling the disease, none are universally safe and effective, and each carries a considerable risk profile. The clinical responses are sometimes typically short duration, and may be accompanied by disease rebound after therapy is withdrawn. All these therapies for psoriasis are only remissive, but not curative. Recently, alternative therapy (such as herbal remedies, nutritional supplements, and dietary manipulation) for the treatment of psoriasis has increasingly received much attention (Traub & Marshall, 2007). Most common reason cited for patients with psoriasis who are interested in alternative therapy was the dissatisfaction with their conventional treatment. Chinese herbal medicines have demonstrated their efficacy in treating psoriasis for hundreds of years. Unfortunately, the use of traditional Chinese herbal medicine, a mixture of various herbs individually formulated for the patient makes scientific analysis extremely difficult. The mechanisms of these herbs were unknown and any possible adverse effect associated these herbs has fallen on dead ears. In this article, we attempt to provide evidence-based information on the clinical benefit of using topical indigo naturalis as a

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mono-therapy for psoriasis and explore the potential mechanisms of indigo naturalis in treating psoriasis. In our experience, the use of topical application can avoid first-pass effect and chemical degradation of the drug in the gastrointestinal tract, as well as minimizing the risk of toxic side effects including gastric irritation, and liver damage. As psoriasis is a persistent and recurring disease, patients often require continuous treatment preferably with a low cumulative toxicity potential. The use of topical application not only presents direct and better therapeutic efficacy but also avoids the long-term adverse reactions. The effects for the use of indigo naturalis have been described in Chinese medical terms as "clearing the heat and cooling the blood" (Lin, 1993). However, the mechanisms underlying the effects of indigo naturalis specially for treating psoriasis seem to be very limited in the published literature. Psoriasis has three principal histological features: epidermal hyperplasia; dilated, prominent blood vessels in the dermis; and an inflammatory infiltrate of leucocytes, predominantly into the dermis (Griffiths & Barker, 2007). Since psoriatic plaques are likely to arise from the failure of normal differentiation process, reagents that suppress hyperproliferation and promote differentiation of epidermal keratinocytes, as well as inhibit inflammation may have a significant impact on the treatment of psoriasis. Our pathological findings verified the above hypothesis that indigo naturalis is indeed effective to improve psoriatic lesion by controlling hyperproliferation and abnormal differentiation of epidermal keratinocytes, as well as lymphocyte infiltration. In addition, as shown in our histological findings, indigo naturalis may have the effect of recovering the epidermal barrier function. In our in vitro study, we were able to demonstrate that the proliferation of epidermal keratinocytes was down-regulated and differentiation was up-regulated after indigo naturalis treatment. Further investigation of pharmacological mechanism of indigo naturalis and its active components in treating psoriasis are currently performed in our laboratory. In general, topical use of indigo naturalis is relatively safe compared to other currently used therapies. However, one should be aware that most ofthe herb indigo naturalis sold in the Taiwan area (this can happen in any area) is not prepared from natural plant. The chemically synthesized indigo naturalis barely contains any effective ingredient, stains the skin and clothing more easily, and tends to induce hypersensitivity. In conclusion, indigo naturalis topically used for treating psoriasis is both safe and effective. We are very pleased to share our experience in the clinic and basic studies of psoriasis treatment which may contribute to the development of promising therapeutic agents for psoriasis.

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ACKNOWLEDGEMENTS Our studies were supported by Grants CMRPG33024 and CMRPG350301 from Chang Gung Memorial Hospital, Taiwan.

REFERENCES Bedi, M.K. and Shenefelt, P.D. (2002). Herbal therapy in dermatology. Archives of Dermatology, 138: 232-242. Griffiths, C.E. and Barker, J.N. (2007). Pathogenesis and clinical features of psoriasis. Lancet, 370: 263-271. Han, J. (1988). Traditional Chinese medicine and the search for new antineoplastic drugs. Journal of Ethnopharmacology, 24: 1-17. Han, R. (1994). Highlight on the studies of anticancer drugs derived from plants in China. Stem cells, 12: 53-63. Hoessel, R., Leclerc, S., Endicott, J.A., Nobel, M.E., Lawrie, A., Tunnah, P., Leost M, Damiens, E., Marie, D., Marko, D., Niederberger, E., Tang, W., Eisenbrand, G. and Meijer, L. (1999). Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclindependent kinases. Nature Cell Biology, 1: 60-67. Knockaert, M., Blondel, M., Bach, S., Leost, M., Elbi, C. and Hager, G.L. (2004). Independent actions on cyclin-dependent kinases and aryl hydrocarbon receptor mediate the antiproliferative effects of indirubins. Oncogene, 23: 4400-4412. Koo, J. and Arain, S. (1998). Traditional Chinese medicine for the treatment of dermatologic disorders. Archieves of Dermatology, 134: 1388-1393. Koo, J. and Desai, R. (2003). Traditional Chinese medicine in dermatology. Dermatologic Therapy, 16: 98-105. Lebwohl, M. (2003). Psoriasis. Lancet, 361: 1197-1204. Lin, X.R. (1993). Psoriasis in China. The Journal of Dermatology, 20: 746-755. Lin, Y.K., Yen, H.R., Wong, W.R., Yang, X.H. and Pang, J.H. (2006). Successful treatment of pediatric psoriasis with indigo naturalis composite ointment. Pediatric Dermatology, 23: 507-510. Lin, Y.K, Wong, W.R. and Pang, J.H. (2007). Successful treatment of recalcitrant psoriasis with indigo naturalis ointment. Clinical and Experimental Dermatology, 32: 99-100. Lin, Y.K, Wong, W.R., Chang, Y.C., Chang, C.J., Tsay, P.K, Chang, S.C. and Pang, J.H. (2007). The efficacy and safety of topically applied indigo naturalis ointment in patients with plaque-type psoriasis. Dermatology, 214: 155-16I. Lin, Y.K, Chang, C.J., Chang, Y.C., Wong, W.R., Chang, S.C. and Pang, J.H. (2008). Clinical assessment of patients with recalcitrant psoriasis in a randimized, observer-blind, vehiclecontrolled trial using indigo naturalis. Archives of Dermatology, 144: 1457-1464. Lin, Y.K., Leu, Y.L., Yang, S.H., Chen, H.W., Wang, C.T. and Pang, J.H. (2009). Antipsoriatic effects of indigo naturlis on the proliferation and differentiation of Kerotinozytes with indirubin as the active component. Journal of Dermatoloued Science, 54: 168-174. Marko, D., Schatzle, S., Friedel, A., Genzlinger, A., Zankle, H., Meijer, L. and Eisenbrand, G. (2001). Inhibition of cyclin-dependent kinasel (CDKl) by indirubin derivatives in human tumor cells. Brit~sh Journal of Cancer, 84: 283-289. Nam, S., Buettner, R., Turkson, J., Kim, D., Cheng, J.Q., Muehlbeyer, S., Hippe, F., Vatter, S., Merz, KH., Eisenbrand, G. and Jove, R. (2005). Indirubin derivatives inhibit State signaling and induce apoptosis in human cancer cells. Proceedings of the National Academy of Sciences to the United States of Aermica, 102: 5998-6003. Ortonne, J.P. (1996). Aetiology and pathogenesis of psoriasis. The British Journal of Dermatology, 135: 1-5. Sreepriya, M., Devaki, T. and Nayeem, M. (2001). Protective effects of Indigofera tinctoria L. against D-Galactosamine and carbon tetrachloride challenge on 'in situ' perfused rat liver. Indian Journal of Physiology and Pharmacology, 45: 428-434.

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Suzuki, K, Adachi, R., Hirayama, A., Watanabe, H., Otani, S., Watanabe, Y. and Kasahara, T. (2005). Indirubin, a Chinese anti-leukaemia drug, promotes neutrophilic differentiation of human myelocytic leukaemia HL-60 cells. British Journal Haematology, 130: 681-690. Traub, M. and Marshall, K (2007). Psoriasis-pathophysiology, conventional, and alternative approaches to treatment. Alternative Medicine ReUlew, 12: 319-330. Tse, T.W. (2003). Use of common Chinese herbs in the treatment of psoriasis. Clinical and Experimental Dermatology, 28: 469-75. Verucchi, G., Calza, L., Attard, L. and Chiodo, F. (2002). Acute hepatitis induced by traditional Chinese herbs used in the treatment of psoriasis. Journal of Gastroenterology and Hepatology, 17: 1342-1343. Xiao, Z., Hao, Y., Liu, B. and Qian, L. (2002). Indirubin and meisoindigo in the treatment of chronic myelogenous leukemia in China. Leukemia & Lymphoma, 43: 1763-17688. Yip, S.Y. (1984). The prevalence of psoriasis in the Mongoloid race. Journal of the American Academy of Dermatology, 10: 965-968. Yuan, Z.Z., Yuan, X. and Xu, Z.X. (1982). Studies on tabellae indigo naturalis in treatment of psoriasis. Journal of Traditional Chinese Medicine, 2: 306.

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3 Development of Anti-Angiogenesis Therapies for Treating Cancer from Chinese Medicinal Herbs STEPHEN

M. SAGAR l ,*, RAlMOND K. WONG l

AND DONALD R.YANCE JR.2

ABSTRACT A new pharmacological approach for managing cancer should simultaneously target the multiple biochemical and physiologic pathways that promote cancer progression. Complex mixtures ofphytochemicals used at relatively low doses can reduce tumor adaptation and resistance to conventional cytotoxic chemotherapy, as well as the prevention ofprogression of microscopic disease following definitive therapy. Angiogenesis is a key process in the promotion of cancer. Many plant-derived chemical mixtures manifest both antiangiogenic as well as other anticancer activities. The present article focuses on products that have a high degree of antiangiogenic activity, but it also briefly describes some of the many other actions of these agents that can inhibit tumor progression and reduce the risk of metastases. Natural health products target molecular pathways other than angiogenesis, including epidermal growth factor receptor, the HER2/neu gene, the cyclooxygenase- 2 enzyme, the nuclear factor kappa-B transcription factor, the protein kinases, the Bcl-2 protein, and coagulation pathways. Antiangiogenic Chinese medicinal herbs include Artemisia annua (Chinese wormwood), Curcuma longa (curcumin), Scutellaria baicalensis (Chinese skullcap), Magnolia officinalis (Chinese magnolia tree), Camellia sinensis (green tea), Ginkgo biloba, Poria cocos, Zingiber officinalis (ginger), Panax ginseng, and Rabdosia rubescens hora (Rabdosia). Quality assurance of appropriate extracts is essential prior to embarking upon clinical trials. More data are required on dose-response, appropriate combinations, and 1. Associate Professor, Departments of Oncology and Medicine, McMaster University,

Canada. 2. Center for Natural Healing, Ashland, Oregon. * Corresponding author: E-mail: stephen.saga~cc.hhsc.ca.

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potential toxicities. Given the multiple effects of these agents, their future use for cancer therapy probably lies in synergistic combinations. During active cancer therapy, they should generally be evaluated in combination with chemotherapy and radiation. In this role, they act as modifiers of biologic response or as adaptogens, potentially enhancing the efficacy of the conventional therapies. Key words : Angiogenesis, anti angiogenic, natural health products, herbal medicine, anticancer, molecular biology

INTRODUCTION The Importance of Antiangiogenesis Therapies for Preventing Cancer Progression The induction and promotion of cancer is a multistep process that involves biochemical interactions from the level of the genes, through cell-signaling pathways, intercellular communication mechanisms, supply of nutrients, channels for metastases, and a host immune response. Part of this process is the induction of new blood vessels to supply nutrients and immunocytes. Many natural health products that inhibit angiogenesis also manifest other anticancer activities. This multi-targeted approach may have major advantages for the treatment of cancer by overcoming the therapeutic resistance to the single drug approach caused by multiple redundant physiological pathways in the cancer cell. Cancer progression requires a source of nutrition and oxygen. Tumors that outgrow their oxygen supply cannot form masses more than 1 to 2 mm in size or they then develop central necrosis. Neoplasms are genetically plastic and often adapt by switching on genes that result in an increased ability to invade and to metastasize. A critical part of this process is the induction of local small blood vessels, termed angiogenesis (Fidler, 2000a; Fidler, 2000b). Tumors do not grow progressively unless they induce a blood supply from the surrounding stroma. Cancers that lack angiogenesis remain dormant. Rapid logarithmic growth follows the acquisition of a blood supply. The tumor angiogenic switch is activated (Fig 1) when the balance of angiogenic inhibitors to stimulators is shifted (Wallace, 2002). The process of neovascularization is subtly controlled in normal tissues by a series of endogenous polypeptides that are secreted during growth, healing, and tissue renewal (Table 1). Cancers synthesize or induce some of these polypeptides, especially vascular endothelial growth factor (VEGF) and angiopoietin (APN). These are peptides that are stimulated by hypoxia and result in sprouting of endothelial cords. The cancer induces a profuse but immature network of thin endothelial-lined channels, essential for tumor oxygenation. Although these new vessels allow progressive tumor growth, they are less efficient than the vascular supply of normal tissues.

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APN normally recruits pericytes and initiates modeling of the vessel wall to more mature forms. However, tumors secrete a relative excess ofVEGF, and this results in disorganized and leaky vessels that cause local bleeding and edema. Antiangiogenic therapy is less susceptible to the development of treatment resistance because it is directed to stromal tissue rather than the genomically unstable tumor cells. Table 1. Endogenous angiogenic polypeptides • • • • • • • • • • • • • • • • • • •

Angiogenin (AG) and Angiotropin (AT) Basic Fibroblast Growth Factor (bFGF) Granulocyte-Colony-Stimulating Factor (G-CSF) Hepatocyte Growth Factor (HGF) Interleukin-8 (11-8) Placental Growth Factor (PGF) Platelet-Derived Endothelial Cell Growth Factor (PD-ECGF) Pleiotrophin (PTN) Proliferin Transforming Growth Factor-a (TGF-a) Transforming Growth Factor-13 (TGF-13) Tumour Necrosis Factor-a (TNF-a) Vascular Endothelial Growth Factor (VEGF) Vascular Permeability Factor (VPF) Insulin-Like Growth Factor I and II (IGF-l and II) Cyclooxygenase (COX) and Lipoxygenase (LOX) Nuclear Factor-Kappa 13 (NF-KB) Activator Protein (AP-l) Angiopoietin (APN)

• Tumour angiogenic switch is activated when balance of angiogenic inhibitors to stimulators is shifted • Reduced production of thrombospondin (TSP-l) from fibroblasts • Vascular endothelial factor (VEGF) stimulates new vessel formation but exceeds Angiopoietin (APN) that normally induces differentiation (pericytes) • Matrix metalloproteinase induction • Increased VEGF relative to APN results in disorganized leaky vessels • Rapid logarithmic growth follows the acquisition of a blood supply

Fig 1. The angiogenic switch

Single antiangiogenic agents, such as monoclonal antibodies or specific tyrosine kinase inhibitors, seem to have limited efficacy. Natural health products contain a range of complex organic chemicals that have synergistic activity. They inhibit angiogenesis by interacting with multiple pathways, as well as having other activities that can interact with cell signaling, the apoptotic pathway, and the interaction of cancer cells with the immune system. Some antiangiogenic agents also have anticoagulation activity that may also be associated with a reduction of metastases. Heparin is a well known example of a therapy with both anticoagulation and antiangiogenic activities. Instead of developing multiple monoclonal antibodies to target

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the various peptides and their receptors, an alternative approach would be to evaluate phytochemicals that influence multiple pathways. The science of pharmacognosy evaluates drugs derived from herbal remedies or phytomedicines. There has been minimal clinical research that evaluates their use as adjuvant therapy to conventional treatment with cytotoxic drugs and radiotherapy. Antiangiogenic natural health products may be most effective in impeding cancer recurrence after cytotoxic therapy, encouraging tumors to remain dormant by changing the balance from cell proliferation to cell death by apoptosis.

The Angiogenic-Metastatic Pathway as a Target for Anticancer Therapies The process of cancer metastasis consists of a series of sequential interrelated steps. Each step is rate limited and may be a target for therapy. The outcome of the process is dependent on both the intrinsic properties of the tumor cells and the responses of the host. The balance of these interactions varies between tumors and patients. The major steps in the formation of a metastasis are as follows (Sugarbaker, 1979; Hart et al., 1989; Liotta & Stetler-Stevenson, 1991): 1. Transformation of normal cells into tumor cells followed by growth. Initially depends on nutrients supplied by simple diffusion. 2. Extensive vascularization (angiogenesis). This must occur if the tumor mass is to exceed 1 mm in diameter. The production and secretion of proangiogenic factors by tumor cells and host cells play a major role in establishing a capillary network from the surrounding host tissue. 3. Local invasion. Tumor cells invade the host stroma through several mechanisms. Thin-walled venules, fragmented arterioles, and lymphatic channels offer little resistance to penetration and provide the most common pathways for tumor cell entry into the circulation. 4. Detachment and embolization. Single cells or clumps break away. Most circulating tumor cells are rapidly destroyed. Those that survive must arrest in the capillary beds of distant organs by adhering either to capillary endothelial cells or to the exposed subendothelial basement membrane. 5. Extravasation into new host organ or tissue. 6. Proliferation within the new host organ or tissue. To continue growing beyond the size of 1 mm in diameter, the micrometastasis must develop a vascular network and evade destruction by host defenses. The cells can then continue to invade blood vessels, enter the circulation, and produce additional metastases. The growth of many cancers is associated with the absence of the endogenous inhibitors of angiogenesis, such as interferon-beta (INF -~). INF-

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~ is a potent inhibitor of angiogenesis through blocking interleukin-8 (IL8), basic fibroblast growth factor (bFGF), and collagenase type V, which are all potent angiogenic factors that aid tumor development and invasiveness. VEGF stimulates the proliferation and migration of endothelial cells and induces the expression ofmetalloproteinases and plasminogen activity. Over expression of VEGF in tumor cells enhances tumor growth and metastasis in several animal models by stimulating vascularization (Folkman & Kotran, 1976; Senger et al., 1983; Folkman et al., 1986; Folkman & Klagsbrun, 1987; Nagy et al., 1989; Folkman et al., 1995; Ferrera et al., 1996; Risau, 1997; Kumar et al., 1998). Some cytotoxic chemotherapy agents are being used at lower than normal doses with the intent of inhibiting angiogenesis and minimizing toxicity (Oliff et al., 1996; Miller et al., 2001). This strategy may permit advanced cancer patients to maintain a better quality of life. The low-dose therapy is termed metronomic dosing (Hanahan et al., 2000; Maraveyas et al., 2005; Hudis, 2005). The metronomic model of conventional cytotoxic chemotherapy suggests that there may also be advantages for administering combinations of phytochemicals that interact with the multistep process of angiogenesis (Singh et al., 2003). In other words, targeting the vascular endothelium with continuous low-dose non-cytotoxic therapies may maintain tumor control without excessive toxicity. Their potential role for increasing overall survival (but not necessarily diseasefree survival) and maintaining quality of life requires evaluation in future clinical trials.

Screening Herbs for Antiangiogenic Activity One ofthe first isolated antiangiogenic agents was a phytochemical. Ingber et al. (1990) reported the antiangiogenic properties of fumagillin, a secreted antibiotic of the fungus Aspergillus fumigatus (Trichocomaceae). Refined fumagillin produces excess toxicity, so analogues of fumagillin were subsequently synthesized. Fumagillin and an analogue labeled TNP-470 are proposed to inhibit angiogenesis by selective inhibition of methionine aminopeptidase type 2 (MetAP-2). However, TNP-470 also demonstrated poor pharmacokinetic behavior and dose-limiting toxicity in clinical trials, and these factors remain obstacles to its use as an anticancer agent. Further modifications of fumagillin have been conducted to develop MetAP-2 inhibitors with desirable pharmacological properties. They have been tested only by in vitro assays, and to date, no clinical trials of these analogues have yet been conducted (Ingber et al., 1990; Pyun et al., 2004; Furness et al., 2005). Since the angiogenic cascade is a multistep process, numerous assays have been developed to study potential angiogenic activity. Some analyze a single step in the pathway, whereas others test the angiogenic cascade as a whole. The relationship of each assay to clinical activity is poorly defined. Some agents have profound anti angiogenic effects at low doses; others exhibit anti angiogenic activity only at near cytotoxic concentrations. Some agents have activity in one model but none in others.

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Criteria for anti angiogenic activity are listed in Fig 4 and should include (Miller et al., 2001): • differential cytotoxicity, • alteration of endothelial cell function, • critical mechanistic effects, and • inhibition of angiogenesis in vivo. Western Biomedicine

Traditional Chinese Medicine • Stagnation of blood and Qi

• Destagnation herbs

• Increased fluid content and abnormal vascular supply; blood stasis; poor oxygenation • Anticoagulants as adjunct to chemotherapy increase survival in clinical studies; prevent blood-borne metastases in animal models

Fig 2. The traditional chinese medicine versus the biomedical theory of cancer

Various assays (Table 2) are used to screen natural health products for antiangiogenic activity (Miller et al., 2001; Wang et al., 2004; Kruger et al., 2001). Assays used for screening will be briefly discussed. Table 2. Screening for anti-angiogenesis

• In vitro Assays - Human umbilical vein endothelial cells (HUVEC) - Bovine aortic endothelial cells (BAEC) - Tumour endothelial cells (TEC) • In vivo Assays - Chick embryo chorioallantoic membrane (CAM) model - Cornea implantation - Matrigel - Tumour xenografts

In vitro Assays In vitro assays are designed to recapitulate each of the multiple events that constitute the angiogenic process. Some of them are very specific in analyzing a single event (proliferation, apoptosis, migration, production of proteases), whereas others provide a more complex picture of the process involving cell functions and interactions with the environment. In vitro assays for the activity of anti angiogenic compounds are usually based on the use of endothelial cells. A critical issue in setting up an in vitro assay is the choice of endothelial cells. Immortalized endothelial cells are sometimes used, as they provide an "unlimited" source of cells. Although these cell lines have the obvious advantages of being easy to grow and relatively stable

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throughout in vitro passages and among batches, they have usually lost some of the characteristics of endothelial cells, including molecular markers, and exhibit changes in function. The most commonly used endothelial cells are from the human umbilical vein, since these are easily available and cell isolation is relatively simple. For the same reasons, bovine or murine aortic endothelial cells are often used too, but these come from large vessels, and they have different phenotypic and behavioral characteristics from those of the microvessels that are more likely involved in angiogenesis. Other common sources of microvascular endothelial cells are the skin, brain, adipose tissue, and adrenal gland. Endothelial cells derived from the microvasculature of different tissues/organs are often heterogeneous, imposing a further constraint on the choice of cell model. Ideally, when developing inhibitors of tumor angiogenesis, tumor-derived endothelial cells should be used. However, practical difficulties in their isolation from tumor tissue and maintenance in culture have limited their use in preclinical studies (Taraboletti et al., 2004). The ability to maintain endothelial cells in culture has allowed the study of endothelial cell proliferation, migration, and cellular function. Angiogenic activity may be represented as endothelial cell migration across a Boyden chamber. Compounds with antiangiogenic potential will inhibit the migration. The bovine aortic endothelial cell (BAEC) and the human umbilical vein endothelial cell (HUVEC) assays are established systems. In vitro assays are relatively inexpensive and give more rapid results. However, the ability to inhibit endothelial cell proliferation, migration, and tubule formation in vitro may not necessarily predict in vivo response. In vitro assays are a rapid method for initial screening of large numbers of agents. Definitive conclusions cannot be based on in vitro assays alone.

In vivo Assays These biological assays are more specific for detecting antiangiogenic activity. The chick embryo chorioallantoic membrane (CAM) model is an extraembryonic membrane that is commonly used to study agents that influence angiogenesis. An angiogenic response occurs 72 to 96 h after stimulation in the form of increased vessel density around the implant. On the other hand, an angiostatic compound induces the vessels to become less dense around the implant and even disappear. Other systems include animal cornea implantation, disc angiogenesis, Matrigel systems, and tumor xenograft models. The in vivo assays provide a more complete physiologic assessment of angiogenesis but are more time-consuming and expensive.

Criteria for Antiangiogenic Activity The degree of anti angiogenic activity is dose dependent. Most chemotherapy drugs have antiangiogenic activity when administered at high doses. We

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are especially interested in compounds that specifically interact and antagonize the steps involved in angiogenesis when administered at low doses. These agents may have relatively low toxicity at low dose and may exhibit a higher therapeutic gain. Most conventional chemotherapy drugs have some degree of anti angiogenic activity as a consequence of their cytotoxic activity. Ideal botanical derivatives would specifically antagonize new vessel formation in tumors, without significant toxicity to normal tissues and without major adverse reactions. The ideal agent would also inhibit tumor cell proliferation through other physiologic pathways, such as influencing intracellular signaling pathways. Multiple levels of antiangiogenic activity may be required to overcome the development of resistance by tumor-associated endothelial cells (TEC). Survival factors, such as the increased secretion of VEGF and bFGF by the tumor cells, activate intracellular pathways that prevent TEC apoptosis. Maximal anti angiogenic activity usually requires prolonged exposure to low concentrations ofthe active agent. This approach contrasts with the concept of administering maximum tolerated doses of cytotoxic drugs to maximize tumor cell kill. Some reports have confirmed the utility of combining low, frequent-dose chemotherapy plus an agent that specifically targets the endothelial cell compartment (Hanahan et al., 2000; Maraveyas et al., 2005). The evidence suggests that an anti angiogenic schedule can be more effective than using high-dose cytotoxic drugs alone. We hypothesize that concomitant scheduling of antiangiogenic botanicals with low, frequentdose cytotoxic therapies may have biological advantages that can increase therapeutic gain.

Natural Health Products that Inhibit Angiogenesis Further research is necessary to screen herbs that may be useful anti angiogenic therapies. Table 3 lists Chinese herbs with anti angiogenic activity (Wang et al., 2004), and Table 4 lists herbs and their derivatives that inhibit VEGF (Singh et al., 2003). An expert herbalist can advise on potential herbal treatments derived from advanced traditional medical systems, such as traditional Chinese medicine. It will be imperative to develop a new model of modern pharmacology based on traditional pharmacognosy. Our developing knowledge of cancer biology suggests that administering cytotoxic drug therapy at very high doses is not always appropriate. A new approach is to administer lower doses of synergistic organic chemicals. These complexes already exist in myriad botanicals. New laboratory techniques allow more specific assays of activity and enable quality assurance and consistency between batches of botanical preparations to be maintained. This will enable credible clinical trials of antiangiogenic natural health products to be initiated.

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Table 3. Antiangiogenesis activity of chinese medicinal herbal extracts (Exhibiting more than 20% inhibition at 0.2 g/herb/mL) (Wang et al., 2004) Assays: chick embryo chorioallantoic membrane (CAM) and bovine aortic endothelial cells culture models (BAEC) Used Part

Name

Berberis paraspecta Catharanthus roseus Coptis chinensis Scrophularia ningpoensis Scutellaria baicalensis Polygonum cuspidatum Taxus chinensis

% Inhibition

% Inhibition

(CAM)

(BAEC)

25 27 25 20 27

38 30 37 34 41 28

Root Leaf Rhizome Root Root Whole plant Bark

26

Traditional Chinese Medical (TCM) theory describes tumor formation as stagnation of qi and blood (Fig 2). In Western theory, this corresponds to the abnormal vasculature induced by the cancer when it induces the angiogenic switch. The tumor has a stagnant blood supply with abnormal leaky vessels that raises the interstitial fluid pressure, resulting in centrifugal diffusion and poor oxygenation. TCM theory suggests "destagnation herbs" as a potential therapy. A randomized placebocontrolled trial from China showed that the addition of "de stagnation" herbs (including Salvia miltiorrhiza Bunge [Lamiaceael and Angelica sinensis Diels [Apiaceae]) to radiotherapy doubled both the local control and survival rates of patients with nasopharyngeal cancer (Fig 3) (Xu et al., 1989). In some clinical trials, anticoagulation drugs are associated with a reduction in metastases (Hejna et al., 1999; Smorenburg et al., 2001; Blom et al., 2005). Laboratory evidence now suggests that many of these Chinese herbs have anti angiogenic and anticoagulation properties (Huang et al., 2003; Wang et al., 2004; Samuels, 2005).

Destagnation herbs (Huo Xue Hua Yu) activate blood flow and improve micro-circulation (huang qi, chi shao, chuan xiong, dang gui, tao ren, hong hua, ji xue teng, ge gen, chen pi, dan shen) N XRT alone XRT + herbs

90 98

Local recurrence

5 Year survival

29% 14% (p<0.05)

37% 53% (p<0.05)

Fig 3. A randomized controlled trial of chinese herb de stagnation formula with radiation in the treatment of nasopharyngeal carcinoma (Xu et al., 1989)

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Some herbs that are traditionally used in China as anticancer agents have been screened for their antiangiogenic activity (Fig 4) in the laboratory (Wang et ai., 2004). Table 3 lists the most active herbs (exhibiting more than 20% inhibition at 0.2 g/herb/mL), using the CAM and BAEC assays. Other herbs are included in Table 4 and are discussed in more detail. Table 4. Chinese herbs and their derivatives that specifically inhibit vascular endothelial growth factor and have direct activity against angiogenesis

• Artemisia annua (Chinese wormwood; contains 95% artemisinin and other related terpenes and flavonoids) • Scutellaria baicalensis (Chinese Baical skullcap; contains 95% baicalin and flavonoids) • Curcuma Zonga (turmeric; contains 95% curcumin) • Camellia sinensis (green tea; contains 95% phenols; 50% epigallocatechin) • Angelica sinensis (Dong quai; contains 4-hydroxyderricin) • Taxus brevifolia (Pacific yew; contains taxol) • Polygonum cuspidatum (Japanese knotweed; contains 20% resveratrol) • Silybum marianum (Milk thistle; contains 80% silymarin [silibin]) • Ginkgo biloba • Magnolia obovata (contains 90% honokiol) • Zingiber officinale (contains 6-gingerol) • Poria cocus • Panax ginseng • Rabdosia rubescens (Rabdosia) • Berberis paraspecta Ahrendt (Berberidaceae), root • Catharanthus roseus G Don (Apocynaceae), leaf • Coptis chinensis Franch (Ranunculaceae), rhizome • Scrophularia ningpoensis Hemsl (Scrophulariaceae), root • Polygonum cuspidatum, Whole plant • Taxus chinensis Rehder (Taxaceae) • Degree of antiangiogenic activity is dose dependent • Specifically interacts and antagonizes angiogenesis at low doses (high therapeutic gain) • Inhibits tumor cell proliferation through multiple physiologic routes, such as intracellular signaling pathways • Minimal effect on normal angiogenesis • Low toxicity with prolonged administration Fig 4. Criteria for an anti-angiogenic botanical

Botanicals usually act on multiple anticancer targets since they contain a variety of organic chemical complexes. The biochemical signaling pathways of angiogenesis form a complex, interconnected web. Inhibition of one part of this web may result in compensation through another pathway. A potential advantage of phytochemicals is that they may act through multiple pathways and reduce the development of resistance by cancer cells. This model of pharmacognosy recognizes the advantage of administering the whole plant product to maximize activity (Fig 5). Over extraction of a specific chemical constituent may remove this therapeutic gain. The

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challenge for modern pharmacognosy is to ensure that the optimum mixture of chemical constituents is maintained when purifying the product. Usually, this will require a combination of both chemical and biological assays. Further anticancer properties of some anti angiogenic botanicals will briefly be discussed. Their effects may also interact with various biochemical pathways that indirectly influence angiogenesis. Traditional practice has been to combine multiple natural health products, and this may scientifically provide a therapeutic advantage. • Range of complex organic chemicals that may have synergistic activity, alone or in combinations. • Inhibit angiogenesis by interacting with multiple pathways. • Bonus activities: interact with cell signaling, apoptosis and interaction of cancer cells with the immune system. • Anticoagulation activity that may be associated with a reduction of metastases • Less acquired resistance to therapy. • Low toxicity: suitable for combination therapy and long term administration. • Possible economic advantages. Fig 5. Advantages of anti-angiogenic NHPs

Chinese Medicinal Botanicals for Potential Development into AntiAngiogenic Therapeutic Agents for the Treatment of Cancer

Artemisia annua (Chinese Wormwood) Artemisinin is the active constituent extracted from the plant A. annua L. (Asteraceae). It has been used clinically as an antimalarial drug (Mueller et al., 2004). More recently, it was shown to be cytotoxic to cancer cells through induction of apoptosis (Singh & Lai, 2004). Artesunate (ART) is a semisynthetic derivative of artemisinin. The in vitro effect of ART was tested on the HUVEC model of angiogenesis. It significantly inhibited angiogenesis in a dose-dependent manner. The inhibition of HUVEC proliferation was greater than the effect on cancer cells, fibroblast cells, and human endometrial cells. This indicates that its anti angiogenic activity is greater than its cytotoxicity. The antiangiogenic effect in vivo was evaluated in nude mice using transplanted human ovarian cancer (HO891) cells and immunohistochemical staining for microvessel CD31 antigen, VEGF, and the VEGF receptor (KDRlflk-1). Tumor growth was decreased and microvessel density was reduced without any toxicity to the host animals. Artemisinin also lowered the VEGF expression by tumor cells and the KDRlflk-1 expression by endothelial cells (Chen & Chang, 2004). Artemisinin also has therapeutic effects through other pathways. It inhibits the activation of nuclear factor-KB (NF-KB), an important activator protein in cancer development and progression (Aldieri et al., 2003).

Scutellaria baicalensis (Chinese Skullcap) Baicalin and baicalein are the main derivatives from the Chinese Skullcap herb, S. baicalensis Georgi (Lamiaceae). They are potent anti angiogenic

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compounds that reduce VEGF, bFGF, 12-lipoxygenase activity, and MMP (Liu et aZ., 2003; Miocinovic et aZ., 2005). It is a component of the traditional Bupleurum formula for stagnation (Xiao Chai Hu Tang): • Bupleurum (Chai Hu) 12-15 g BupZeurum chinense • Scutellaria (Huang Qin) 9-12 g Scutellaria baicaZensis • Pinelli a (Ban Xia) 9-12 g Pinellia ternata • Fresh Ginger (Sheng Jiang) 3-6 g Zingiberis offici naZis • Ginseng (Ren Shen) 6-9 g Panax ginseng • Licorice (Gan Cao) 3-6 g GZycyrrhiza uraZensis • Jujube Dates (Da Zao) 3-5 pcs Zizyphus jujuba S. baicaZensis is also one of the herbs found in PC-SPES, a complex of Chinese herbs that may have clinical activity against advanced prostate cancer (Small et aZ., 2000; Oh et aZ., 2001). However, although a phase 2 trial demonstrated clinical activity in patients with androgen independent prostate cancer, diethylstilbestrol (DES) and ethinyl estradiol (both known to have potent antiprostate cancer activity) were detected in various lots of PC-SPES (Hsieh et aZ., 2002). It is still intriguing that the decline in PSA was greater for PC-SPES that was potentially contaminated with DES than for the comparator group that received DES alone, suggesting some independent activity. Although baicalin and baicalein have multiple anticancer activities in vitro, their clinical activity is not established, and their contribution to any potential therapeutic effect of PC-SPES is unknown (Cordell, 2002; Oh et aZ., 2004).

Curcuma longa (Turmeric) Curcumin is the most active curcuminoid present in turmeric, C. Zonga L. (Zingiberaceae). It interacts with cancer cells at a number of levels and can enhance the tumoricidal efficacy of cytotoxic chemotherapy and radiotherapy (Narayan, 2004; Khafif et aZ., 2005; Sen et aZ., 2005). Its antiinvasive effects are partly mediated through the down regulation of MMP2 and the upregulation of tissue inhibitor of metalloproteinase (TIMP-l) (Shao et aZ., 2002). These enzymes are involved in the regulation of tumor cell invasion. Curcumin inhibits the transcription of 2 major angiogenesis factors, VEGF and bFGF (Arbiser et aZ., 1998). It interacts with VEGF and nitric oxide- mediated angiogenesis in tumors (Sreejayan, 1997; GarciaCardena et aZ., 1998). Elevated levels of nitric oxide correlate with tumor growth. Curcumin reduces nitric oxide generation in endothelial cells. CD13/ aminopeptidase-N (APN) is a membrane bound enzyme found in blood vessels undergoing active angiogenesis. Curcumin binds to APN and blocks its activity, thereby inhibiting angiogenesis and tumor cell invasion (Gururaj et aZ., 2002; Shim et aZ., 2003). Derivatives of curcumin may be

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developed to target APN, providing a novel approach to reduce neoplastic angiogenesis (John et al., 2002; Hahm et al., 2004). Curcumin also downregulates the expression of the VEGF and MMP-9 genes that are associated with angiogenesis. Demethoxycurcumin is a structural analogue of curcumin isolated from Curcuma aromatica Salisb. (Zingiberaceae). It specifically inhibits the expression of MMP-9 (Kim et al., 2002). Curcumin can interfere with the activity of both MMP-2 and MMP-9, the basis of the angiogenic switch, thereby reducing the degradation of the extracellular matrix (Chen et al., 2004). It also interferes with the release of angiogenic factors that are stored in the extracellular matrix. It inhibits growth factor receptors such as epidermal growth factor receptor (EGFR) and VEGFR and the intracellular signaling tyrosine kinases. This cell-signaling system can promote further angiogenesis through gene activation that increases levels of cyclooxygenase-2 (COX-2), VEGF, IL-8, and the MMPs (Reddy & Aggarwal, 1994; Dorai et al., 2001; Leu et al., 2003). A phase 1 study of curcumin found no treatment-related toxicity up to 8000 mg/d. Beyond 8000 mg/d, the bulky volume of the drug was unacceptable to the patients. The serum concentration of curcumin usually peaked at 1 to 2 h after oral intake of curcumin and gradually declined within 12 h (Cheng et al., 2001). This study suggested that it may prevent cancer progression. Derivatives of curcumin, such as copper chelates of curcuminoids, may have increased antitumor activity (John et al., 2002).

Camellia sinensis (Green Tea) C. sinensis (L.) Kuntze (Theaceae), contains polyphenols and catechins (mainly epigallocatechin-3 gallate [EGCG]) (Lee et al., 2002). These constituents inhibit MDAMB breast cancer cell and HUVEC proliferation (Sartippour et al., 2002). In addition, they suppress breast cancer xenograft growth and reduce the density of tumor vessels in rodent studies (Cao & Cao, 1999). This is associated with a decrease in VEGF, regulated at the level of transcription. EGCG also suppresses protein kinase C (PKC), another VEGF transcription modulator. Inhibition of VEGF transcription is one of the molecular mechanisms involved in the anti angiogenic effects of green tea that may contribute to its potential use for cancer treatment (Kojima-Yuasa et al., 2003; Tang et al., 2003; Fassina et al., 2004). EGCG may be administered as a powdered extract of green tea. An appropriate dose has been extrapolated from antiangiogenic activity in rodent experiments as well as a phase 1 study in humans (Cao et al., 1999; Pisters et al., 2001). A dose of 1.0 g/m3 times daily (equivalent to 7-8 Japanese cups [120 mLl 3 times daily) has been recommended. In practice, lower total daily doses of 2 to 4 g of standardized green tea extract (95% polyphenols/60% catechins) are usually prescribed. Each gram of this extract provides 400 to 500 mg of EGCG. The dose-limiting adverse effects are gastrointestinal and

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neurological effects of caffeine. However, the caffeine may potentiate the antiangiogenic effect of EGCG (Pisters et al., 2001).

Polygonum cuspidatum (Japanese Knotweed) This contains 20% resveratrol. It has anti angiogenic activity demonstrated by its ability to inhibit HUVEC division and to decrease the lytic activity of MMP-2 (Cao et al., 2005). Resveratrol inhibits VEGF induced angiogenesis by disruption of reactive oxygen species-dependent Src kinase activation and subsequent VE-cadherin tyrosine phosphorylation (Igura et al., 2001; Lin et al., 2003). Resveratrol inhibits the growth of gliomas in rats by suppressing angiogenesis (Tseng et al., 2004).

Silybum marianum (Milk Thistle) Silibinin and silymarin are polyphenolic flavonoids isolated from the fruits or seeds of S. marianum (L.) Gaertn. (Asteraceae). In the laboratory, silymarin demonstrates strong activity against a variety of tumors through down regulating VEGF and EGFR (Jiang et al., 2002; Singh & Aggarwal, 2003). Silymarin suppresses VEGF when used as a single agent against human ovarian cancer endothelial cells in vitro (Gallo et al., 2003). A more detailed discussion can be found elsewhere (Sagar, 2007).

Ginkgo biloba G. biloba L. (Ginkgoaceae) extract has anticancer effects that are related to its gene-regulatory and antiangiogenic properties. The G. biloba extract used in most of the research is EGb 761, which contains about 25% flavonoids (ginkgo-flavone glycosides) and about 5% terpenoids (ginkgolides and bilobalides). The most potent flavonoid is ginkgolide B. This extract inhibits angiogenesis by down regulating VEGF (Zhang et al., 2002; De Feudis et al., 2003).

Magnolia officinalis (Chinese Magnolia Tree) The seed cones of M. officinalis Rehder & E.H. Wilson (Magnoliaceae) contain substances that inhibit the growth of new blood vessels. Honokiol is the active constituent. It may partly reduce angiogenesis through the regulation of platelet-derived endothelial cell growth factor and TGF-f3 expression. It also inhibits nitric oxide synthesis and TNF-f3 expression (Son et al., 2000; Lee et al., 2004). In animal experiments, it suppresses the proliferation of blood vessel endothelial cells more than other types of cells and thereby reduces tumor growth (Bai et al., 2000; Chen et al., 2004).

Zingiber officinale (Ginger) 6-Gingerol, fromZ. officinale Roscoe (Zingiberaceae), inhibits both the VEGFand bFGF-induced proliferation of human endothelial cells and causes cell cycle arrest. It also blocks capillary-like tube formation by endothelial cells in response to VEGF and strongly inhibits sprouting of endothelial cells in

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the rat aorta and mouse cornea in vitro models. In mice receiving injections of B16F10 melanoma cells, intraperitoneal administration of 6-gingerol, at doses less than cytotoxic levels, reduces the number of lung metastases (Kim et al., 2005).

Poria cocos P. cocos F.A. Wolff (Coriolaceae) is a mushroom extract that has been traditionally held to have anticancer activity. It inhibits platelet aggregation

and appears to be antiangiogenic by downregulating NF-kappa-B (Jin et al., 2003; Chen et al., 2004; Lee et al., 2004).

Panax ginseng The lipophilic constituents of P. ginseng C.A. Meyer (Araliaceae) are called saponins (or ginsenosides). These extracts possess anticancer activities in tumors that include antiangiogenesis and induction of tumor cell apoptosis (Sato et al., 1994).

Rabdosia rubescens (Rabdosia) The herb R. rubescens H . Hara (Lamiaceae) is used traditionally to treat cancer and is a constituent of the PC-SPES formula. It contains ponicidin and oridonin, two diterpenoids that possess significant antiangiogenic activity (Meade-Tollin et al., 2004).

CONCLUSIONS Angiogenesis involves multiple interdependent processes operating at the molecular level. These include gene expression, signal processing, and enzyme activities. Most antiangiogenic natural health products block new vessel formation at multiple levels. Lack of standardization of screening assays may be an obstacle to defining the most effective products for clinical use. Over extraction of constituents may negate some of the advantages of potential synergy. Mainly preclinical data exist for most of the naturally derived anti angiogenic agents. Most of the studies of anti angiogenic activity are based on in vitro or animal work, which cannot be readily extrapolated to humans. Phase 1 and 2 studies are required to determine their potential to improve cytotoxic therapies. Quality assurance of appropriate extracts is essential prior to embarking on clinical trials (Fig 6). Since anti angiogenic agents are mainly cytostatic in nature, the usual paradigm for anticancer drug development, in which tumor response in phase 2 trials prompts further development, is not always appropriate. More data are required on dose response, appropriate combinations, and potential toxicities. Given the multiple effects of these agents, their future use for cancer therapy probably lies in synergistic combinations. They may

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4·

wi

Traditional Qbs~tvati!:>nS'

Authentication ;lmf;f~oslational research

Dose optimization

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effi l'ldverse taifects

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be evaluated alone for the prevention of cancer recurrence following definitive treatment. To be suitable for long-term chronic use, these agents should possess minimal toxicity and should be orally administered. However, angiogenesis is also essential for healing of injuries. Most compounds that inhibit tumor angiogenesis are likely to inhibit physiologic angiogenesis, leading to potential side effects, such as ulceration and bleeding. Studies are required to determine distinguishing features of tumor vessels from normal vessels to enable a therapeutic gain to be achieved. During active cancer therapy, they should generally be evaluated in combination with chemotherapy and radiation. In this role, they act as biological response modifiers and adaptogens, potentially enhancing the efficacy of the so-called conventional therapies. The diversity of angiogenic factor expression in different tumors receiving various therapies, combined with the fact that endothelial cells in different tumors are phenotypically distinct, is a major challenge for the development of effective antiangiogenic regimens (Jung et at., 2000; Sweeney et at., 2003). Their effectiveness may be increased when multiple agents are used in optimal combinations. Surrogate markers, such as angiogenic cytokines, are necessary to predict anti angiogenic response. Circulating levels of FGF-2, VEGF, vascular adhesion molecule (V-CAM-1), endothelial intercellular adhesion molecule (ICAM-l), IGF-1, and cytokines such as IL-8 may correlate with tumor angiogenesis (Yoshida et at., 1997; Tang et at., 2001; Brower, 2003; Ruegg, et at., 2003; Ria et ai., 2004). In addition, circulating endothelial cells and their progenitors may be a more reliable marker of response to anti angiogenic therapies (Salcedo et at., 2005; Schneider et ai., 2005; Shaked et ai., 2005). Noninvasive functional imaging, such as positron emission tomography and functional magnetic resonance imaging, may playa role (Neeman, 2000).

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Current laboratory evidence suggests a useful role for natural health products in the treatment of cancer. The input of an ethnobotanist, oncologist, laboratory scientist, and a clinical trials methodologist to the research effort is essential to distill the wealth of traditional knowledge into a modern framework that can be evaluated scientifically. The determination of the biologically active dose that may possess least toxicity may be relevant. Combinations of whole herbs or constituent phytochemicals at lower doses are important. In addition, a longer period of exposure to the natural health product may be more effective than a short exposure to the highest possible dose level. New designs for trials to demonstrate activity in human subjects are required. Although controlled trials might be preferred, smaller studies with appropriate end points and surrogate markers for anti angiogenic response could help prioritize agents for the larger resource-intensive phase 3 trials. Because most of the agents are expected to be cytostatic, it is inappropriate to require the standard criteria of measured tumor response. On the other hand, simply confirming stable disease may be misleading. More research on surrogate markers of antiangiogenic response is obviously necessary prior to directing resources to large-scale clinical trials. A multidisciplinary approach by the herbalist and the oncologist is important for implementing and studying natural health products used for the treatment of patients with cancer. We now have a better understanding of their effects at the molecular level. Introducing these interventions into the clinic through appropriate studies will provide more definitive evidence of efficacy and hopefully improve outcome for many cancer patients.

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Miller, KD., Sweeney, C.J. and Sledge, G.W. (2001). Redefining the target: chemotherapeutics as antiantiogenics. J Clin Oncol., 19: 1195-1206. Miocinovic, R, McCabe, N.P. and Keck, RW. (2005). In vivo and in vitro effect of baicalein on human prostate cancer cells. Int J Oncol., 26: 241-246. Mizushina, Y., Akihisa, T. and Ukiya, M. (2004). A novel DNA topoisomerase inhibitor: dehydroebriconic acid; one of the lanostane-type triterpene acids from Poria cocos. Cancer Sci., 95: 354-360. Mueller, M.S., Runyambo, N. and Wagner, I. (2004). Randomized controlled trial of a traditional preparation of Artemisia annua L. (Annual Wormwood) in the treatment of malaria. Trans R Soc Trop Med Hyg., 98: 318-321. Nagy, J.A., Brown, L.F. and Senger, D.R (1989). Pathogenesis of tumor stroma generation: a critical role for leaky blood vessels and fibrin deposition. Bioch~m Biophys Acta, 948: 305-326. Narayan, S. (2004). Curcumin, a multi-functional chemopreventive agent, blocks growth of colon cancer cells by targeting betacatenin-mediated transactivation and cell-cell adhesion pathways. J Mol H~stol., 35: 301-307. Neeman, M. (2000). Preclinical MRI experience in imaging angiogenesis. Cancer Metastasis Rev., 19: 39-43. Oh, W.K, George, D.J. and Hackmann, K (2001). Activity of the herbal combination, PCSPES, in the treatment of patients with androgen-independent prostate cancer. Urology, 57: 122-126. Oh, W.K, Kantoff, P.W. and Weinberg, V. (2004). Prospective, multicenter randomized phase II trial of the herbal supplement: PC-SPES, and diethylstibestrol in patients with androgen- independent prostate cancer. J Clin Oncol., 22: 3705-3712. Oliff, A., Gibbs, J.B. and McCormick, F. (1996). New molecular targets for cancer therapy. Sci Am., 275: 144-149. Pisters, KM.W., Newman, RA. and Coldman, B. (2001). Phase I trial of oral green tea extract in adult patients with solid tumors. J Clin Oncol., 19: 1830-1838. Pyun, H.J., Fardis, M. and Tario, J. (2004). Investigation of novel fumagillin analogues as angiogenesis inhibitors. Bworg Med Chem Lett., 14: 91-94. Reddy, S. and Aggarwal, B.B. (1994). Curcumin is a non-competitive and selective inhibitor of phosphorylase kinase. Fed Eur Bwchem Soc Lett., 341: 19-22. Ria, R, Portaluri, M. and Russo, F. (2004). Serum levels of angiogenic cytokines decrease after antineoplastic radiotherapy. Cancer Lett., 216: 103-107. Risau, W. (1997). Mechanisms of angiogenesis. Nature, 386: 671-674. Ruegg, C., Meuwly, J.-Y. and Driscoll, R. (2003). The quest for surrogate markers of angiogenesis: a paradigm for translational research in tumor angiogenesis and anti angiogenesis trials. Curr Mol Med., 3: 673-691. Sagar, S.M. (2007). Future directions for research on Silybum marianum for cancer patients. Integrat Cancer Ther., 6: 166-173. Salcedo, X., Medina, J. and Sanz-Cameno, P. (2005). Review article: angiogenesis soluble factors as liver disease markers. Al~ment Pharmacol Ther., 22: 23-30. Samuels, N. (2005). Herbal remedies and anticoagulant therapy. Thromb Haemost., 93: 3-7. Sartippour, M.R, Shao, Z.M. and Heber, D. (2002). Green tea inhibits vascular endothelial growth factor (VEGF) induction in human breast cancer cells. J Nutr., 132: 2307-2311. Sato, K, Mochizuki, M. and Saiki, I. (1994). Inhibition of tumor angiogenesis and metastasis by a saponin of Panax ginseng, ginsenoside-Rb2. Bioi Pharm Bull., 17: 635-639. Schneider, M., Tjwa, M. and Carmelie, P. (2005). A surrogate marker to monitor angiogenesis at last. Cancer Cell., 7: 3-4. Sen, S., Sharma, H. and Singh, N. (2005). Curcumin enhances vinorelbine mediated apoptosis in NSCLC cells by the mitochondrial pathway. Biochem Biophys Res Commun., 331: 1245-1252. Senger, D.R., Galli, S.J. and Dvorak, A.M. (1983). Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science, 219: 983-985. Shaked, Y., Bertolini, F. and Man, S. (2005). Genetic heterogeneity of the vasculogenic phenotype parallels angiogenesis: implications for cellular surrogate marker analysis of antiangiogenesis. Cancer Cell, 7: 101-111.

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4 An Anthology of the Studies on the AntiInflammatory, Antinociceptive and Febrifuge Effects of Medicinal Plants in Turkey ESRA KUPELI AKKOL 1 AND ERDEM YESlLADA2

ABSTRACT Inflammatory diseases are among the most common health problems treated with traditional remedies. Therefore it is crucial to evaluate the potential of herbal remedies for the discovery of novel bioactive compounds that might serve as leads for the development of potent drugs. Although several agents are known to treat these types of disorders, prolonged use should be avoided due to severe side effects. Consequently, there is a need to develop safe and new anti-inflammatory agents with minimum side effects. In this context, traditional medicines provide a vast source for the discovery of original drug leads. The present investigation represents a preliminary screening in an ongoing program on plants used in Turkish traditional medicine for the treatment of rheumatism and related inflammatory diseases. In this study, selected plants as the subject of the present study were recorded in the alphabetical order of plant families. Key words : Antinociceptive, anti-inflammatory, bioassay-guided processing, ethnopharmacology, febrifuge, folk remedies, plants, Turkey 1. Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler 06330, Ankara, Turkey. 2. Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, Kayisdagi 34755 Istanbul-Turkey. * Corresponding author: E-mail: [email protected]

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INTRODUCTION Acute and chronic inflammations are known to be complicated processes induced by several different classes of chemical mediators, e.g. prostaglandins, leukotrienes and platelet-activating factor, etc. Antiinflammatory agents exert their effect through a spectrum of different modes of action (Samuelsson et al., 1978). However, chronic inflammatory diseases are still one of the major health problems worldwide and nonsteroid anti-inflammatory drugs (NSAIDs) are the most prescribed drugs for treatment of inflammatory diseases. Although the NSAIDs provide the patients with symptomatic relief, they do not modify the pathogenesis of inflammation and do not reduce the disabling bone and cartilage damage (Ford-Hutchinson et al., 1981). Therefore, it has become a must to search for new initiatives in the treatment of chronic inflammation. On the other hand, plants have been used by human being since ages in traditional medicine due to their therapeutic potential and the search on medicinal plants have led the discovery of novel drug candidates used against diverse diseases. The plant kingdom encompasses a wide range of plants that possess biologically active molecules of medicinal value. Many such plant-derived molecules have been isolated, identified, and successfully introduced into international markets by pharmaceutical industries that are mostly located in the developed world. In contrast, plant-based remedies, prescribed by folk doctors in the developing world for the treatment of many diseases, remain largely restricted in their use to the local communities in which such remedies were developed (Sumner, 2000). Although such plant-based remedies have recently received considerable interest from the medical and pharmaceutical industries, only a small fraction of such plants have been assessed. In the past few years, many studies have investigated the healing potential of higher plants with ethnobotanical histories. This stemmed from several reasons such as consumer interest in such plantbased remedies, the concern about the possible side effects of allopathic medicine, the lower cost of phytotherapy, and the fact that many plantbased or herbal remedies succeeded in replacing allopathic medicines in relieving malady symptoms. The aim of this manuscript is to review the literature of bioactive anti-inflammatory plant extracts and their isolated active components as an addition to the literature typically used in folk medicine in Turkey basin to treat inflammation related ailments.

ANACARDIACEAE The nuts of Pistacia vera L. (Anacardiaceae), commonly referred to pistachio, is a quite popular flavoring foodstuff and snack in Turkey and in the world. The oleoresin of P. vera, a widely-distributed tree throughout

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the south-east region of Anatolia, has been used to treat asthma by chewing as folk remedy in this part of Turkey as well as against stomachache (by chewing the gum) and hemorrhoids (externally) (Baytop, 1999). In the light oftraditional information, the ethanolic and aqueous extracts prepared from different parts (leaf, branch, fruit, stalk) of Pistacia vera as well as its oleoresin were evaluated for their in vivo anti-inflammatory and antinociceptive activities (Orhan et al., 2006). Only the oleoresin, out of the all extracts screened herein, exhibited a dose-dependent anti-inflammatory activity on carrageenan-induced hind paw edema model in mice ranging between 27.4-34.7% at 250 mg/kg and 32.6-38.7% at 500 mg/kg doses without inducing any gastric damage. The potency of oleoresin at 500 mg/kg dose was almost equal to that observed for indomethacin, a reference agent. As for antinociceptive activity, the oleoresin also displayed 32.1 % inhibition at 500 mg/kg dose onp-benzoquinone induced abdominal contractions in mice, while inhibitory rate decreased to 21.7% at 250 mg/kg. Fractionation of the oleoresin indicated that n-hexane fraction to be active, which further led to recognition of some monoterpenes, mainly a-pinene (77.5%) by capillary gas chromatography-mass spectrometry (GC-MS) as well as the oleoresin itself. a-Pinene was also assessed for its antinociceptive and antiinflammatory activities in the same manner and exerted a moderate antiinflammatory effect at 500 mg/kg dose. APIACEAE In Turkish folk medicine various species of the Eryngium are used for a wide range of ailments; particularly roots are used against various inflammatory disorders, oedema, sinusitis, urinary infections or inflammations etc. and snake or scorpion bites or goiter, roots and leaves for infertility and herbs for wound healing as well as food while fresh (Sezik et al., 1997; Yesilada & Sezik, 2003). Eryngium species, particularly E. campestre and E. maritimum are called in Turkish folk-medicine as "Bogadikeni" and widely distrubuted in all parts of Turkey. Infusion of aerial parts and roots of these species are used in folk remedies as antitussive, diuretic, appetizer, stimulant and aphrodisiac (Baytop, 1999). Aqueous extract ofthe roots of E. campestre is used orally and poultice is applied externally as a remedy aganst scorpion bite in the Balikesir-Turkey (personal interview). Similar utilizations were found in the old documents from the medieval ages such as against wound and burns, pains, hemorrhoids and sexual diseases (Lev, 2002). Effects of the roots from different Eryngium species against various inflammatory conditions are also well-known in the traditional medicines worldwide; for example, E. campestre as anti-oedema (Leporatti & Ivancheva, 2003) or E. creticum for inflammatory wounds (AliShtayeh et al., 1998). It is also documented in the popular phytotherapy books for such treatments, i.e. E. campestre is recommended against kidney and urinary tract inflammations and oedema (Gruenwald et al. 2000a). To

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confirm the traditional usage ethanolic and aqueous extracts obtained from either aerial parts or roots of E. campestre, E. creticum, E. davisii, E. falcatum, E. isairicum, E. kotschyi, E. maritimum, and E. trisectum were evaluated for their in vivo anti-inflammatory and antinociceptive activities (Kiipeli et al., 2006a). According to the results of investigations, except E. falcatum extracts, ethanol extracts either from the aerial parts or roots of Eryngium species showed apparent anti-inflammatory activity on carrageenan-induced hind paw oedema and TPA-induced ear oedema tests and antinociceptive activity on p-benzoquinone-induced writhing test. In spite of potent activity of the ethanol extract from E. isairicum aerial parts was induced gastric damage. Aerial parts and roots of E. maritimum and E. kotschyii were found to possess most promising activities without including any apparent gastric damage. Moreover, Yesilada et al. (1989) reported that the methanol extract (150 mg/kg) from the whole plant of E. billardieri showed a 44% inhibition on increased vascular permeability induced by acetic acid in mice on oral administration. Further studies were conducted on the various solvent fractions obtained from the aerial parts and roots of the plants and they were tested for their effects on increased vascular permeability induced by acetic acid model, as well as serotonin, bradykinin, and carrageenan-induced paw oedemas in mice. A significant antiinflammatory activity was observed only in the precipitated part of the nbutanol extract of the roots, which mainly contains saponins (Yesilada et al., 1989).

Seseli is an old Greek name that was called by Hippocrates for certain members of the Apiaceae family (Hamlyn, 1969). The genus Seseli L., is composed of aromatic herbs and economically important species that are used as foods, spices, condiments and ornamentals (Lawrence, 1969; Crowden et al., 1969; Pimenov & Leonov, 1993). Several Seseli species are reported in ancient literature for various healing effects. The roots of S. mairei Wolff., have been used against inflammations, swelling, rheumatism, pain and common cold in Chinese traditional medicine (Hu et al., 1990). The seeds of an Indian species, S. indicum, have been reported to possess anthelmintic, carminative, stomachic and stimulant properties (Tandan et al., 1990). In Turkish folk medicine, the fruit of S. tortuosum is used as emmenagogue and anti-flatulence (Baytop, 1999), while the leaves of S. libanotis (Kelemkesir or Kelemenkesir in Turkish) are consumed as a vegetable in the eastern Turkey (Baytop, 1999). Traditional usage of the genus in folk medicine led us to search other species for their biological activies. The ethyl acetate and methanol (80%) extracts obtained from ten Seseli L. species (Apiaceae) growing in Turkey, Seseli andronakii Woron., S. campestre Besser, S. gummiferum Pall. ex Sm. subsp. corymbosum (Boiss & Heldr) P. H. Davis, S. gummiferum Pall. ex Sm. subsp. gummiferum, S. hartvigii Parolly & Nordt, S. libanotis (L.) W. Koch, S. petraeum M. Bieb., S. peucedanoides (Bieb.) Koso-Pol., S. resinosum Freyn & Sint., S. tortuosum

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L. were evaluated for their in vivo anti-inflammatory and antinociceptive activities (Kiipeli et al., 2006c). Among the plant extracts, the ethyl acetate extracts from S. andronakii, S. campestre, S. gummiferum subsp. corymbosum, S. petraeum, S. resinosum and S. tortuosum showed 30.1%, 32.3%, 36.9%, 39.8%, 35.1%, 37.6% inhibition in p-benzoquinone-induced abdominal constriction test, respectively. The ethyl acetate extracts of S. gummiferum subsp. corymbosum, S. petraeum, S. resinosum and S. tortuosum also exhibited notable inhibition, ranging between 24.5-29.7%, 28.1-33.3%, 17.4-27.5%, 27.9-31.3%, respectively, in carrageenan-induced hind paw edema model at 100 mg/kg dose without inducing any gastric damage. During the acute toxicity evaluation, neither death nor gastric bleeding was observed for any of the plant extracts. Results have supported the traditional use of some Seseli species against inflammatory disorders. APOCYNACEAE

Nerium oleander L., oleander, is widely distributed in the Mediterranean region. Due to the toxic nature, limited number of utilization have been recorded in Turkish folk medicine. Among them a speculative assertion on cancer treatment by a Turkish medical doctor became very popular and eventuated recently the leaf extract undergoing Phase I clinical evaluation as a potential treatment against cancer in USA (Smith et al., 2001). During our expeditions, an informant described that fresh oleander flowers was put in alcohol in summer and kept as a home-remedy to alleviate her severe pain and paralysis in her legs. In a reference survey in the database program of Turkish folk medicine (TUHIB), a similar utilization was also encountered by another informant, i.e. flowers kept 40 days in olive oil was applied on joint against rheumatic pain (Yesilada, 2002). Moreover, leaves or flowers are used to stop pain or eczema, sap obtained from fresh leaves for abscess or rheumatism were recorded. Besides, another part of oleander, the fruits, is reported as antirheumatic and as a remedy for skin diseases in Saudi folk medicine (Adam et al., 2001). In this context, Zia et al. (1995) reported that two purified fractions (B 1 and B3 ) obtained from the methanol extract of fresh oleander leaves possess a eNS depressant activity, i.e. produced reduction in locomotor activity, rotarod performance and potentiate hexobarbital induced sleeping time. Moreover, they showed significant analgesic activity as indicated by inhibitory effects on acetic acidinduced writhings and increased reaction time to thermal test. Our results also supported that both aqueous and ethanol extracts of oleander leaves possess significant antinociceptive activity as decided by using another writhing test model, but the effect of EtOH extract was more pronounced (Erdemoglu et al., 2003). However, both extracts were shown gastric ulcerogenicity in mice. On the other hand, EtOH extracts from either dried or fresh flowers exhibited potent antinociceptive activity without inducing any gastric damage and potency was more pronounced than that of reference

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drug aspirin. Experimental results have clearly confirmed that liposoluble fractions of flowers, as decided by ethanol or olive oil extracts were used in folk medicine, possess remarkable antinociceptive activity. Antiinflammatory activity assessment experiment has also verified the conclusion that liposoluble components of flowers may have the activity since only the EtOH extracts were found active even than that of reference drug indomethacin.

ASTERACEAE The genus Achillea, named after the mythological Greek warrior Achilles, comprises of approximately 85 species, most of which are endemic to Europe and the Middle East. In the Turkish flora 40 Achillea species have been recorded and 20 of them are endemic (Davis, 1982). On the otherhand, seveal Achillea species have been known to be used in folk remedies for various purposes such as haemorrhoid and wound healing (Baytop, 1999). Especially, A. millefolium is frequently used against diarrhoea, abdominal pain and stomachache in Turkish traditional medicine (Yesilada et al., 1993; Fujita et al., 1995; Honda et al., 1996). In order to evaluate their folkloric utilization, both antinociceptive and anti-inflammatory activities of five Achillea species including Achillea wilhelmsii C. Koch,A. setacea Waldst & Kit, A. vermicularis Trin., A. phrygia Boiss. & Bal. and A. sipikorensis Hausskn. et Bornm. were investigated (Kiipeli et al., 2007a). The ethanol extracts of A. wilhelmsii, A. setacea and A. vermicularis showed significant antinociceptive (p-benzoquinone-induced writhing test) and antiinflammatory (carrageenan-induced hind paw edema model) activity at 500 mg/kg dose, per os, without inducing any apparent acute toxicity as well as gastric damage. A. phrygia was shown to possess only significant antinociceptive activity, while A. sipikorensis did not show any remarkable anti-inflammatory and antinociceptive activity. Furthermore, the essential oils obtained by water distillation from aerial parts of Achillea schischkinii Sosn. and Achillea aleppica DC. subsp. aleppica were analysed by GC and GC/MS. 1,8-Cineole (32.5% and 26.1%, respectively) was the main component in both oils. Their antimicrobial, anti-inflammatory and antinociceptive activities were also studied and the oil of A. aleppica subsp. aleppica showed significant anti-inflammatory, antinociceptive and moderate antimicrobial activities. The oil of Achillea aleppica subsp. aleppica contains 6.6% of a-bisabolol and its derivatives. These compounds might be at least partially responsible for antiinflammatory activity of the oil (Iscan et al., 2006). In folk medicine, Helichrysum species have been used for gall bladder disorders as medicinal tea, because of their bile regulatory and diuretic effects (Siizge~ et al., 2005). Some of them are used for anti-inflammatory and anti-allergic properties (Carini et al., 2001; Tepe et al., 2005). Moreover,

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this genus is traditionally used in the treatment of wounds, infections and respiratory conditions (Lourens et al., 2004). In Turkey, several Helichrysum species are used in folk medicine as diuretic, cholagogue, and against kidney stones (Baytop, 1999). In order to evaluate the anti-inflammatory and antinociceptive activities of Helichrysum species (Kiipeli et al., 2006b), aqueous extracts from H. arenarium (L.) Moench. subsp. aucheri (Boiss.) Davis & Kupicha and H. plica tum DC. subsp. plicatum were investigated and they showed 37.0%, 30.4% inhibition at 100 mg/kg doses and 31.2%, 28.3% at 200 mg/kg doses in p-benzoquinone-induced abdominal constriction test, respectively, without inducing any gastric damage. The aqueous extracts of all the plants did not show any anti-inflammatory activity in carrageenaninduced hind paw edema model at 50, 100 and 200 mg/kg doses. During the acute toxicity evaluation, neither death nor gastric bleeding was observed for any of the plant extracts.

BERBERIDACEAE Roots and barks of various Berberis species are used as folk remedy for the treatment of various inflammatory diseases such as lumbago, rheumatism and to reduce fever. In Semerkand Bazaar (Uzbekistan) a black tough material prescribed for the treatment of lumbago attracted our attention during our scientific expeditions. The herb dealer described that it was the concentrated aqueous extract of Berberis obionga roots. In a reference survey, several other Berberis species were also found to be used in the treatment of various inflammatory conditions, including rheumatism, fever and pyrexia. In order to evaluate this information, the effect of a widespread Turkish species, B. crataegina DC. was studied and found that the roots of the plant possessed significant anti-inflammatory, analgesic and febrifuge effects. Through bioassay-guided fractionation and isolation procedures berberine was isolated as the main active ingredient responsible from the relevant effects. In that paper, however, it was addressed that the role of other alkaloids in the effect of the roots should also be investigated (Yesilada & Kiipeli, 2002). In the study of Kosar et al. (1999) three types ofisoquinoline alkaloids were detected in the roots, barks and branches of Turkish Berberis species: protoberberine (berberine, palmatine,jatrorrhizine, columbamine), bisbenzylisoquinoline (berbamine, oxyacanthine, aromoline) and aporphine (magnoflorine) types. In a following study, the effects of these alkaloids were studied using various in vivo models in mice (Kiipeli et al., 2002). All alkaloids inhibited inflammations in varying degrees, among them berberine, berbamine and palmatine were shown to possess significant and dosedependent inhibitory activity against serotonin-induced hind paw oedema both on oral and topical applications and acetic acid-induced increase in vascular permeability on oral administration. Moreover, these three alkaloids were also shown to possess dose-dependent antinociceptive activity,

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which assessed by using the model based on the inhibition of p-benzoquinoneinduced writhing movements as well as antipyretic activity on FCA-induced increased rectal temperature on subacute administration. However, all alkaloids induced gastric lesions in varying degrees (Kiipeli et al., 2002). Since it is well known that anti-inflammatory agents acting through inhibition of cyclooxygenase-l (COX-I) enzyme induce such gastric ulceration, the effect of isoquinoline alkaloids might also be based on this mechanism. As a matter of fact, berberine was found to inhibit castor oilinduced diarrhoea in rats, which supported this conclusion (Yesilada & Kiipeli, 2002). Fukuda et al. (1999) demonstrated that berberine inhibits cyclooxygenase-2 (COX-2) transcriptional activity in colon cancer cells in a dose-dependent manner, however, the effect on constitutive enzyme (COX1) was not determined. It is noteworthy that powdered roots of B. lycium is suggested to be taken with milk for the treatment of rheumatism and muscular pains in Pakistan folk medicine, probably to protect the gastric mucosa from damage (Ikram et al., 1966). The results of the previous study (Kiipeli et al., 2002) revealed that berberine, berbamine and palmatine were the main anti-inflammatory, antinociceptive and antipyretic alkaloids in the roots of Berberis species. Especially the effect of first two was more pronounced. Since alkaloid composition ofthe roots showed great variations from species to species, the relevant activity would highly be species-specific. Thus, utilization of B. integerrima roots in therapy would not be beneficial due to the higher concentrations of the alkaloids with weak activity, i.e. magnoflorine and oxyacanthine. On the other hand, due to the higher concentration of effective alkaloids in B. cretica and B. vulgaris roots, more beneficial effects would be expected. Berberine and related isoquinoline alkaloids have been reported to exhibit a broad distribution in the plant kingdom (Keys, 1985), such as; Epimedium sp. leaves (Berberidaceae); Phellodendron sp. barks and Xanthophyllum sp. roots (Rutaceae); Coptis sp. rhizomes and Hydrastis sp. roots (Ranunculaceae); Sanguinaria sp. rhizomes (Papaveraceae); etc. In traditional medicine, some of these plants are known to be employed in the treatment of various inflammatory ailments and results of the present study would also be helpful to explain the relevant effects as well as safety evaluation of these plant remedies especially regarding to the ulcerogenic effect of these alkaloids reported in that study (Kiipeli et al., 2002). Contrary to the observations of the present study that berberine type alkaloids induce remarkable gastric lesions, berberine was reported to be effective in the treatment of gastritis and gastric ulcers in some documents (PDR, 2000), which needs further studies and explanations.

BRASSICACEAE In Turkish folk medicine, Isatis sp. is known to be used for wound healing and against constipation (Baytop, 1999). In Iran, another species of the

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plant, 1. cappadocia, has also been reported against rheumatism, asthma, eczema, fever, headache and wound healing. Rezaeipoor et al. (2000) showed that the plant possess in vivo suppressive effect on humoral primary immune response at the dose of 250 mg/kg, while stimulated secondary immune responses at the dose of 500 mg/kg. Additionally, the activity of a Turkish species, Isatis glauca Aucherx Boiss. ssp. glauca, was studied and ethanol extract demonstrated a significant antinociceptive activity (37.2%) at 500 mg/kg, and a medium anti-inflammatory activity (24-28% inhibition) (Kupeli et al., 2007c).

CISTACEAE In Turkish folk medicine, the leaves of the Cistus laurifolius are also used externally as an effective remedy against several inflammatory ailments such as rheumatic pain, high fever and urinary inflammations. For relieving rheumatic pain a warm decoction of leaves is used as a bath or wilted leaves are externally applied onjoints. For alleviation or treating of urinary inflammations, a poultice is prepared by boiling the leaves and then mixing with flour. This poultice is then applied externally as a plaster on the dorsal part ofthe body in a line with the kidneys (Sezik et al., 1991; Yesilada et al., 1995). Effects of the extracts and fractions from the leaves with non-woody branches ofCistus laurifolius L. (Cistaceae) were studied using two in vivo models of inflammation in mice. For the preliminary antiinflammatory activity assessment of C. laurifolius leaves two types of extracts (aqueous and ethanolic) were prepared and investigated against carrageenan-induced hind paw oedema and acetic acid-induced increased vascular permeability models in mice. Aqueous extract did not show any remarkable effect against carrageenan-induced hind paw edema model, while EtOH extract was significantly active both in 250 and 500 mg/kg doses. A comparable result was obtained against another inflammatory model based on the inhibition of increased vascular permeability, induced by the intraperitoneally injection of acetic acid. Subsequently, through bioassay-guided fractionation and isolation procedures three flavonoids; 3-0-methylquercetin, 3,7 -O-dimethylquercetin and 3,7-0dimethylkaempferol were isolated as the main active ingredients from the ethanol extract (Fig 1). Moreover, these flavonoids were shown to possess potent antinociceptive activity, which was assessed through inhibition ofp-benzoquinone-induced writhing reflex. Results ofthe present study have clearly supported the utilization of C. laurifolius in Turkish traditional medicine and three flavonoids were shown to have strong antinociceptive and anti-inflammatory activities, per os without inducing any apparent acute toxicity as well as gastric damage (Kiipeli & Yesilada, 2007).

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OH Me O

OH

OH

OH

OH

o

0

OH MeO

OH

0

Fig 1. Chemical structures of active compounds

DIOSCOREACEAE Tamus communis root is used to treat rheumatism after pounded in Turkish folk medicine (Yesilada et al., 1995). In a previous report, the ethanol extract from Tamus communis roots was shown to possess significant inhibitory effect on granuloma formation when tested on cotton pellet granuloma model in rats (Capasso et al., 1983; Mascolo et al., 1987). However, in our study neither aqueous nor ethanol extracts prepared from both roots and aerial parts of that plant did show any remarkable anti-inflammatory or antinociceptive activity (Kupeli et al., 2007 c).

ERICACEAE The sap obtained from a fresh branch of Rhododendron ponticum L. is dropped into the toothcave against toothache in Turkish folk medicine (Yesilada et al., 1999). The flowers and fruits of another Rhododendron species, R. molle, have also been recorded in ancient and modern monographs as analgesic and insecticide in Chinese Traditional Medicine (Li et al., 2000). Moreover, the leaves of R. chrysanthum in Siberia (Grieve, 1994) and R. ferrugineum in Germany (Chosson et al., 1998) have been reported as a remedy against rheumatism. In a reference survey, we did not find any experimental study related to this subject, however, Manjeet and Ghosh (1999) reported that quercetin isolated as a major component of R. cinnabarium inhibited LPS-induced nitric oxide and tumor necrosis factor-production in murine macrophages, which might be related with the aforementioned effects. In vivo studies also confirmed that leaves of the

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plant possess a significant antinociceptive activity. The EtOH extract showed the highest antinociceptive activity among the plants investigated as well as a potent anti-inflammatory activity without inducing any gastric lesion was recorded (Erdemoglu et al., 2003). The genus Erica is represented only four species of which one taxon (E. bocquetti (Pe~men) P. F. Stevens) is endemic in Turkey. Among the other species, E. arborea L. and E. manipuliflora Salisb. are widespread species common in the coastal sides in Turkey, while E. sicula Guss. subsp. libanotica (C. & W. Barbey) P. F. Stevens has a limited distribution in South West Anatolia (Stevens, 1978). Herbal tea prepared from aerials parts of E. arborea or E. manipuliflora has been popularly used as diuretic, astringent and treatment of urinary infections in Turkey (Baytop, 1999; Tuzlaci & Erya~ar Aymaz, 2001). In particular, a glass of 5% infusion or decoction of E. arborea leaves is suggested after meals to discharge the edema from the body in slimming formulations (Ba~er et al., 1986; Antonone et al., 1988). In the light of traditional usage anti-inflammatory and antinociceptive activities of different extracts were prepared with methanol, chloroform, ethyl acetate, n-butanol and water from the aerial parts of E. arborea L., E. manipuliflora Salisb., E. bocquetii (Pesmen) P. F. Stevens and E. sicula Guss. subsp. libanotica (C. & W. Barbey) P. F. Stevens (Ericaceae) of Turkish origin were investigated by using in vivo methods (Kiipeli Akkol et al., 2008c). The ethyl acetate extracts of E. arborea (EAE), E. bocquetii (EBE) andE. manipuliflora (EME) exhibited notable inhibition against carrageenan-induced (24.1-32.3%, 23.8-36.1%, 29.2-35.1%, respectively) and PGE 2 -induced (21.2-37.7%, 6.8-29.7%, and 6.2-34.1%, respectively) hind paw edema as well as TPA-induced mouse ear edema models in mice, while the ethyl acetate extract of E. sicula subsp. libanotica (ESE) (10.7-29.7%) displayed potent anti-inflammatory activity only on the PGE 2-induced hind paw edema model. However, the remaining extracts were found to be inactive against inflammatory models. Same extracts i.e., EAE, EBE and EME were also found to exhibit remarkable antinociceptive activity inp-benzoquinone-induced abdominal constriction test at a dose of 100 mg/kg (46.5%,27.7% and 36.3%, respectively) (Kiipeli Akkol et al., 2008c). The anti-inflammatory and antinociceptive activities were mainly determined in the ethyl acetate extracts. Previous studies have shown that flavonoids, anthocyanoids, coumarins, terpenic compounds especially ursolic acid and essential oils were present in Erica species (Vieitez & Ballester, 1972; Ballester et al., 1975; Bennini et al., 1993; Chulia et al., 1995; Dufor et al., 2007). Flavonoids exert significant scavenging properties on oxygen radicals and can interfere with the production of arachidonic acid metabolites trough lipoxygenase enzyme inhibition and reduce the concentration of leukotrienes (Reyes Ruiz et al., 1996; Halliwell & Chirica 1993).

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Calluna vulgaris L. (heather), which is also known as "the true heather of Europe", is the sole member ofthe genus Calluna (Ericaceae) in Turkey (Baytop, 1999). Heather has a long history of medicinal use in folk medicine. In Anatolia, infusion ofthe plant has been used as urinary disinfectant and diuretic (Baytop, 1999). Different ethnobotanical uses ofthe plant have been documented as antiseptic, astringent, cholagogue, depurative, diaphoretic, diuretic, expectorant, mildly sedative, vasoconstrictor and antirheumatic as well as for the treatment of gout symptoms in European societies (Mabey, 1979; Chiej, 1984; Kumarasamy et al., 2002). A homeopathic remedy is prepared from the fresh branches is prescribed in the treatment of rheumatism, arthritis and insomnia (Launert, 1981). The plant is also suggested among the Bach flower remedies and the keywords for prescribing it are "self-centeredness" and "self-concern" (Chancellor, 1985). In order to evaluate this ethnobotanical information, its anti-inflammatory and antinociceptive activities were studied using in vivo experimental models in mice (Orhan et al., 2007b). The ethanolic extract of the plant was first fractionated into five extracts; namely, n-hexane, chloroform, ethyl acetate (EtOAc), n-butanol, and water fractions. Among them, the EtOAc fraction was found to be the most effective and was further subjected to bioassayguided fractionation and isolation procedures. After successive column chromatography applications, on Sephadex LH-20 and silica gel, a component, which is responsible for the above-mentioned activities ofthis species of Turkish origin, was isolated and its structure was elucidated as kaempferol-3-0-[3-D-galactoside, a common flavonol derivative by means of spectral techniques (Fig 2) (Orhan et al., 2007b). A reference survey has revealed that in vitro anti-inflammatory activity of C. vulgaris was previously OH HO

o OH

o

;::O~~

'~ HO

Fig 2. Chemical structure ofkaempferol-3-0-j3-D-galactoside

reported by two separate studies. In the first study, the water extracts of 52 plants from 28 different families, where selections were based upon the records for their use in inflammatory diseases and treatment of wounds in Swedish traditional medicine, were investigated for their inhibitory activities on prostaglandin biosynthesis and platelet activating factor (PAF)-induced exocytosis in vitro and was found highly efficient on both assays (Tunon et

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al., 1995). In particular, C. vulgaris was one of the three plants, which exhibited the most potent cyclooxygenase inhibition in that study. In connection to these data, the flowers of C. vulgaris, was tested in vitro for its effect on arachidonic acid metabolism using mouse peritoneal macrophages, human platelets, and differentiated HL60 leukemic cells (N ajid et al., 1992). Among the several organic and aqueous extracts of heather flowers, the acetone extract was found to inhibit lipoxygenase (LOX) enzyme and further analysis of this extract led to the isolation of ursolic acid as an active anti-lipoxygenase component. Ursolic acid was shown a higher efficiency on 5-LOX inhibition than 12- and 15-LOX, whereas it had a 38% inhibition on cyclooxygenase. Many flavonoids, isolated from medicinal herbs used in traditional medicines, are endowed with desired biological activities su~h as antimicrobial, anti-hepatotoxic, anti-inflammatory, cardioprotective, antiosteoporotic, and anti-cancer (Middleton, 1998). Amongst them, quercetin and kaempferol are known to be the most common flavonols present in many plants in different glycosidic forms. In one study, two coumaryl glycosides ofkaempferol (platanoside and tiliroside) were reported to exhibit strong anti proliferative effect suppressing DNA synthesis in a dose- and time-dependant manner (Dimas et al., 2000). In a very good accordance with the above data (Orhan et al., 2007b), Gil et al. (1994) also figured out that kaempferol-3-0-f3-D-galactoside possessed a remarkable antiinflammatory activity on both in vivo carrageenan-induced hind paw edema and 12-0-tetradecanoylphorbol-3-acetate-induced ear edema as well as in vitro inhibitory effect on phospholipase-~ enzyme. In some similar studies, various kaempferol glycosides have also shown to possess significant inhibitory activities on TNF -{X, production and nitric oxide (NOS) release (Liang et al., 1999; Authore et al., 2001; Matsuda et al., 2001).

GENTIANACEAE Isoorientin is a common C-glycosyl flavone, luteolin-6-C glucoside, and has been reported in many different plant species such as Gentiana, Asphodelus, Rumex, Swertia and Vitex species (Harborne, 1994). During the studies on a Turkish folk medicine, Gentiana olivieri Griseb. (Gentianaceae), isoorientin was isolated as the active anti-hepatotoxic component from the flowering herbs through in vivo bioassay-guided fractionation procedures against carbon tetrachloride-induced hepatic damage (Deliorman-Orhan et al., 2003). In a following bioassay-guided activity assessment study on the same plant material same compound was again isolated as the active hypoglycaemic component (Sezik et al., 2004). In a reference survey, several other biological activities were attributed to isoorientin including potent antioxidant (Ko et al., 1998), antimicrobial (Afifi et al., 1997), myolitic activity on smooth musclecontaining preparations from the rat and guinea pig uterus (Afifi et al., 1999), etc. Isoorientin (Fig 3) was also shown to possess significant

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antinociceptive and anti-inflammatory activities without inducing any apparent acute toxicity as well as gastric damage. Isoorientin induced inhibition between 28.4-37.1 % and 36.4-43.4% against carrageenan-induced inflammation at 15 and 30 mg/kg doses, respectively. The most remarkable point was the anti-inflammatory activity of isoorientin was found almost equal to that of indomethacin (10 mg/kg) which exerted 30.4-40.3% activity, in a very close dose level. Maybe more important point was isoorientin did not induce any visible gastric damage. Due to the high gastric lesion risk of nonsteroidal anti-inflammatory agents, i.e. indomethacin and acetyl salicylic acid, this is a very important peculiarity. On the other hand, isoorientin was also found to possess significant antinociceptive activity, though not potent as acetyl salicylic acid (Kiipeli et al., 2004). Literature survey demonstrated that isoorientin possesses a wide range of biological activities. Due to the adjacent hydroxyl groups at ring B, i.e. catechol functions, the most prominent biological activity reported for isoorientin is a potent antioxidant activity (Diena et al., 2003). Isoorientin showed a strong antiperoxidative activity towards linoleic acid (Mun'im et al., 2003). The antioxidant activity ofisoorientin was also reported by Budzianowski et al. (1991) through evaluating the inhibitory effect on MDA concentration stimulated by Fe+3 ions and ascorbic acid in rat liver microsomes. Ko et al. (1998) studied the antioxidant activity ofisoorientin-6"-0-glucoside, a watersoluble form, from Gentiana arisanensis (Gentianaceae) using various models and reported a potent antioxidant and radical scavenging activity and suggested as an antioxidant therapy against a wide range of free radicalinduced disorders particularly to protect human LDL from oxidative attack. Isoorientin was isolated as one of the active anxiolytic ingredients from a Peruvian folk remedy, Jatropha cilliata M. Arg. (Euphorbiaceae) at 40 and 100 mg/kg doses and the activity at the later dose was identical to that of diazepam. Under the same conditions, a similar effect was observed for orientin obtained from another fraction, however luteoline, aglycone of these two flavonoids, was ineffective. In the same work, analgesic effect was also investigated and isoorientin was found to have more potent effect than orientin (Okuyama et al., 1996). H

OH

H OH

H

H

OH

Fig 3. Structure ofisoorientin

H

H

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GERANIACEAE Geranium species are used as antiasthmatic, antiallergic, antioxidant, antidiarrhoeic, antihepatotoxic, diuretic, tonic, haemostatic, stomachic and antidiabetic in folk medicine (Baytop, 1999). G. pratense ssp. finitimum is particularly used to treat stomachache in in Turkey (Ozaydin et al., 2006). As a relevant activity, G. maculatum has been reported to treat duodenal ulcers as well as in diarrhoea and hemorrhoids in Canada (British Herbal Pharmacopeia, 1987). The leaves of G. thunbergii have been used as an antidiarrhoeic in Japan (Fujiki et al., 2003) and G. sanguineum was reported to possess a potent effect against influenza virus (Sokmen et al., 2005). To obtain an experimental evidence on the therapeutic efficacy of Geranium species and its phenolic compounds for inflammatory diseases, the aqueous extract of aerial parts of G. pratense subsp. finitimum (Woronow) Knuth, its fractions and isolated compounds, i.e. the mixture of quercetin 3-0-aR,

OH

HO

OH

1

R}

R2

OH

H H H H H H H OH

H

2

OH OH

3 5

H

6

OH OH OH

0

R3 a-arabinopyranose f3-galactopyranose f3-glucopyranose f3-galactopyranose f3-glucopyranose (2"-O-galloyl)-f3-glucopyranose (2"-O-galloyl)-f3-galactopyranose (2"-O-galloyl)-f3-glucopyranose OH

~

CI

#

OH

'/11111

0 0H

OH

4 (-)-6-chloroepicatechin

Fig 4. The chemical structures ofthe isolated compounds from Geranium pratense subsp. finitimum

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arabinopyranoside, kaempferoI3-0-[3-galactopyranoside (1), the mixture of quercetin 3-0-j3-glucopyranoside, quercetin 3-0-j3-galactopyranoside (2), kaempferoI3-0-j3-glucopyranoside (3), (-)-6-chloroepicatechin (4), the mixture of quercetin 3-0-(2-0-galloyl)-j3-glucopyranoside, quercetin 3-0-(2-0-galloyl)[3-galactopyranoside (5) and myricetin 3-0-(2-0-galloyl)-j3-glucopyranoside (6) (Fig 4) were examined on carrageenan-, PGE 2 - and TPA-induced inflammation in mice and p-benzoquinone induced writhing reflex to assess anti-inflammatory and antinociceptive activities. The effective dose of materials for the inhibition of carragenan-induced hind paw edema assay was determined to be 100 mg/kg, which was also used in the assays with the extract, its fractions and isolated compounds in all other experiments. The aqueous extract as well as compounds 1 and 2 (100 mg/kg) significantly inhibited the formation of the carrageenan-induced hind paw edema. Significant reductions were also exerted by the aqueous extract and the compounds 1,2 and 5 against PGE 2-induced hind paw edema and TPAinduced ear edema models. The same compounds were shown to possess antinociceptive activity (Kupeli et al., 2007 d). JUGLANDACEAE

Juglans regia L. leaves have been used mostly in worldwide traditional medicines as antifungal, antihelmintic, astringent, keratolytic, antidiarrhoeal, hypoglycaemic, depurative, tonic and for the treatment of sinusitis, cold and stomachache (Girzu et al., 1998; Mouhajir et al., 2001). In Turkish folk medicine, however, leaves are frequently used to reduce fever in sunstroke or to alleviate rheumatic pain, externally. Fresh leaves are spread to form a bed for unclothed patient, who is covered with fresh leaves or pounded with/without raw salt and applied on the naked body or forehead to reduce fever or on swelled joint to alleviate the rheumatic pain, or boiled in water and used for bathing in both conditions (Fujita et al., 1995; Ye~ilada, 2002). Both aqueous and EtOH extracts showed significant antinociceptive activity without inducing any gastric damage, but the effect of later extract was more pronounced than the former as well as that of aspirin (Erdemoglu et al., 2003). EtOH extract also showed a potent antiinflammatory activity as potent as indomethacin. Since direct application of fresh leaf is suggested for the treatment, less polar components of the leaves might possibly be more active (Erdemoglu et al., 2003). LAMIACEAE Prunella vulgaris L. is used against rheumatism, colds and cardiac disorders in Turkish folk medicine (Yesilada et al., 1993). In a previous study, a herbal combination (SKI 306X) consisting of Clematis mandshurica, Trichosanthes kirilowii and Prunella vulgaris, which was used in Chinese traditional medicine for the treatment of various inflammatory disorders, i.e. lymphadenitis and arthritis, was studied for their in vitro effects as anti-inflammatory, analgesic,

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antiarthritic, blood microcirculation-enhancer as well as inhibition of cartilage degeneration enzyme activities on proteoglycan degradation in cartilage explant culture and collagenase-induced rabbit ostheoarthritis model (Choi et al., 2002). The results revealed that SKI 306X inhibited proteoglycan degradation in a concentration-dependent manner and it also showed protective effect on the knee joint of rabbit from ostheoarthritis-like changes on prophylactic administration. In another study, the aqueous extract of P. vulgaris was shown to possess strong antioxidant potency in inhibiting rat erythrocyte hemolysis and lipid peroxidation in rat kidney and brain homogenates (Liu & Ng, 2000). On the other hand, in a study was performed by our group, in vivo anti-inflammatory or antinociceptive activity of the plant was found to be weak (Kupeli et al., 2007). The genus Lamium L. (Lamiaceae) is characterized by almost 40 annual or perennial herbaceous plants spread throughout Europe, Asia, and Africa (Willis, 1973). Lamium album, L. maculatum, and L. purpureum exhibit antispasmodic, anti-inflammatory, antioxidant and antiproliferative properties and have been extensively used in folk medicines as a remedy for the treatment of several disorders such as trauma, fractures, putrescence, paralysis, leucorrhoea, hypertension, as well as women affiictions, such as menorrhagia, uterine hemorrhage, vaginal or cervical inflammations (Bremness, 1995; Matkowski & Piotrowska, 2006; Paduch et al., 2006; Shuya et al., 2003; Weiss, 1988). In Turkish flora, thirty Lamium species have been recorded (Duman, 2000a; Mill, 1982). Among them, L. album, L. maculatum, and L. purpureum have been reported to be used as tonics and for the treatment of constipation in Anatolia (Baytop, 1999). In western Anatolia (in particular in Manisa) whole plant of L. album and several other Lamium species are used to relieve pain in rheumatism and other arthritic ailments (Ozaydin et al., 2006). In order to assess the traditional usage various extracts were prepared from several Lamium species. The n-butanol extracts of L. garganicum subsp.laevigatum (LGL-BuOH), L. garganicum subsp. pulchrum (LGP-BuOH), and L. purpureum var. purpureum (LPPBuOH) exhibited notable inhibition (16.5-28.9%, 14.5-26.9%, 12.3-21.5%, resp.) in carrageenan-induced hind paw edema model at doses of200 mg/kg without inducing any gastric damage. The LGL-BuOH (7.1-30.4%) and LGPBuOH (5.9-24.1%) extracts also displayed potent anti-inflammatory activity against PGE 2-induced hind paw edema model. However, the remaining extracts were found to be inactive against carrageenan-induced hind paw edema and PGE 2-induced hind paw edema models. On the other hand, all the extracts of the title plants failed to display any anti-inflammatory activity on TPA-induced mouse ear edema model. LGL-BuOH and LGP-BuOH were also found to exhibit remarkable antinociceptive activity inp-benzoquinoneinduced abdominal constriction test at a dose of200 mg/kg (25.0% and 24.3%, respectively). The experimental data demonstrated that L. garganicum subsp. laevigatum and L. garganicum subsp. pulchrum displayed remarkable anti-inflammatory and antinociceptive activities (Kiipeli Akkol et al., 2008b).

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Salvia species, Sage, features prominently in the national pharmacopoeias of many countries throughout the world and several species have been used as a remedy against perspiration and fevers; as a carminative; a spasmolytic; an antisepticlbactericidal; an astringent; as a gargle or mouthwash against the oral inflammations such as tongue or throat; a wound-healing agent; in skin and hair care; and against rheumatism (Kintizios, 2000). S. virgata is known as "yilancik" in Turkey and used to alleviate dermatological problems and wounds. The decoction of S. virgata is used against blood cancers in Western Turkey (Baytop, 1999). S. halophila is an endemic plant of Turkey and traditionally being used as herbal tea. To provide a scientific evidence for traditional uses, the aerial parts of S. halophila and S. virgata were subjected to Soxhlet extraction with different solvents i.e. n-hexane, ethyl acetate, methanol and aqueous methanol (50%). Plants were also extracted with water under reflux. The effects ofthe extracts were studied inp-benzoquinone-induced abdominal constriction test for the assessment of antinociceptive activity and carrageenan-induced hind paw oedema and 12-0-tetradecanoyl-13acetate (TPA)-induced ear oedema models in mice for assessing the antiinflammatory activity. Results showed that methanol extract significantly inhibited carrageenan-induced paw oedema and p-benzoquinone-induced abdominal constrictions at 100 mglkg dose, while it was ineffective against the TPA-induced ear oedema. However, the other extracts did not show any inhibitory antinociceptive and anti-inflammatory activities on these in vivo models. The extracts were then analysed using a HPLC-PDA method. Rosmarinic acid was found as the main constituent in the extracts, while caffeic acid and luteolin derivatives were also detected in small ratio (Kiipeli Akkol et al., 2008a). In a literature survey, Hosseinzadeh et al. (2003) reported that the aqueous extract of Salvia leriifolia seeds showed significant and dose-dependent (1.25-10 g/kg) antinociceptive activity over 7 h, and the activity was inhibited by naloxone pretreatment in hot-plate and tail flick tests. They also observed significant and dose-dependent (2.510 glkg) activity against acute inflammation induced by acetic acid and in the xylene ear oedema test. In the chronic inflammation test (cotton pellet test), however, the extract (2.5-5 g/kg) demonstrated considerably significant and dose-dependent anti-inflammatory activity. Consequently, a supraspinal antinociceptive effect mediated by opioid receptors has been suggested as the activity mechanism of extract (Hosseinzadeh et al., 2003). On the other hand, in a previous study, the methanol extract of Salvia miltiorrhiza was found to inhibit production ofPGD 2 and the ethyl acetate subfraction exerted the strongest inhibition. From the ethyl acetate subfraction, tanshinone I was isolated as an active principle through activity-guided isolation procedures (Kim et al., 2002). The genus Sideritis L., widely distributed in MediterraneanMacronesian region, are traditionally used in Spanish folk medicine for their anti-inflammatory and gastroprotective properties to treat certain

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disorders that are accompained by inflammation. Several anti-inflammatory compounds have been obtained from the members of this genus, mainly flavonoids and terpenoids (Godoy et al., 2000). In Turkey, this genus is represented by 46 species (Duman, 2000b; Ayta!; & Aksoy, 2000) and some of which are used in the treatment of gastrointestinal ailments, common colds (including bronchitis, sore throat, flu) and as a diuretic as well as a herbal tea for pleasure in Turkish folk medicine (Baytop, 1999; Yesilada et al., 1995). In the light oftraditonal usage, acetone extract from the aerial parts of Sideritis ozturkii Aytac & Aksoy and its fractions were investigated for its in vivo anti-inflammatory and antinociceptive activities. Acetone extract of the plant and its phenolic fraction were found to possess significant inhibitory activity on carrageenan-induced hind paw oedema and pbenzoquinone induced writhing reflex models in mice. Ozturkoside A (chrysoeriol 7-0- [2'"-0-caffeoyl-6'"-O-acetyl-13-D-glucopyranosyl-( 1~2)-13- Dglucopyranoside]) (1), ozturkoside B (chrysoeriol 7-0-[2"'-0-caffeoyl-13-Dglucopyranosyl-(1~2)-13-D-glucopyranoside]) (2) and ozturkoside C (chrysoeriol 7-0- [2'"-0-p-coumaroyl-6'"-O-acetyl-13-D-glucopyranosyl-( 1~2)13-D-glucopyranoside]) (3) were isolated from the active phenolic fraction (Fig 5). Ozturkoside C showed notable antinociceptive and anti-inflammatory activities without inducing any apparent acute toxicity or gastric damage.

OH

OH

HO

0

a

Fig 5. Chemical structures of compounds 1-3

Although the activity of ozturkosides A and B were not significant in statistical analysis, some inhibitory effect was observed. Accordingly, it is suggested that these components in phenolic fraction might possibly share the antinociceptive and anti-inflammatory activities together (Kiipeli et al., 2007b). In spite of a high number of studies reporting the anti-inflammatory and antinociceptive activities of several Sideritis species only two flavonoids have been isolated and defined as the active constituents; hypolaetin-8-

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glucoside (Villar et al., 1984) and 5-0-demethylnobiletin (Bas et al., 2006). As shown in Fig 6, hypolaetin-8-glucoside shows very close chemical structure to ozturkoside C, both having luteoline type flavone glycoside structure. OH OH O-Glu HO

o OH

Fig 6. Chemical structure ofhypolaetin-8-glucoside

Additionally, acetone extract of S. stricta Boiss. & Heldr. apud Bentham and its phenolic fraction exhibited potent inhibitory activity against both bioassay models in mice. From the active phenolic fraction a well-known phenylethanoid glycoside, verb as cos ide (= acteoside) (1) and two flavonoid glycosides, isoscutellarein 7-0- [6'" -O-acetyl-~- D-allopyranosyl(1 ~2)l-~-D-glucopyranoside (2), and isoscutellarein 7-0-[6'" -O-acetyl-~-D­ allopyranosyl-( 1~2)l-6f1-0-acetyl-~- D-glucopyranoside (3) were isolated. During phytochemical studies we also isolated a methoxyflavone, xanthomicrol (4) from the non-polar fraction (Fig 7). Although antinociceptive and anti-inflammatory activities of phenolic components were found not significant in statistical analysis, compounds 1 to 3 showed a notable activity without inducing any apparent acute toxicity as well as gastric damage. Furthermore, a mixture of flavonoid glycosides (2 + 3) exhibited a significant inhibitory effect in both models at a higher dose (Kiipeli et al., 2007c).

HO

HO

1

87

Effects of Medicinal Plants in Turkey Fig 7. Contd. R OH HO

OCOCH3

o o

HO OH

2 R:OH 3 R: OCOCH 3 4'

OH

OH

0 4

Fig 7. Chemical structures of compounds 1 - 4

LAURACEAE Laurus nobilis L., laurel, is a spice plant widely distributed in the Mediterranean area and the seed oil is used against rheumatic pain in Turkish folk medicine as well as in Europe. Previously, the oil obtained from laurel fruit was also reported to be used for treatment of furuncles, sprains, bruises and rheumatism (Simic et al., 2003). The experimental results indicated that the ethanol extract of the seeds possesses a remarkable anti-inflammatory (29.1-37.3%) and antinociceptive (34%) activity at a dose of 500 mg/kg (Kupeli et al., 2007c). On the other hand, extract prepared from the leaves were also studied for comparison and was found completely devoid of any activity (Kupeli et al., 2007 c).

LORANTHACEAE The genus Viscum L. (Loranthaceae) comprises semi-parasitic plants which grow on various host tree and shrubs (Miller, 1982). Viscum album L. is the most widespread species worldwide and has been reputed against cardiovascular diseases, i.e. hypertension and atherosclerosis; various bone and joint disorders including periarthritis, spondylitis, and arthritis; to

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alleviate headache; for immune system stimulation; in nervous disorders as sedative or to combat epilepsy (Bartram, 1995; Murray, 1995; Wichtl & Bisset, 1994). In order to evaluate this information, antinociceptive and anti-inflammatory activity ofthe five flavonoids [5,7 -dimethoxy naringenin (1,3, and 5) or 4', 6'-dimethoxy chalcononaringenin (2, 4)] derivatives; (5,7dimethoxy-flavanone-4' -0- ~- D-gi ucopyranoside (1), 2'-hydroxy-4' ,6'dimethoxy-chalcone-4-0-~- D-glucopyranoside (2), 5,7 -dimethoxy-flavanone4'-0- [2"-0-(5111-0-trans-cinnamoyl)-~-D-apiofuranosyl]-~- D-glucopyranoside (3), 2'-hydroxy-4' ,6'-dimethoxy-chalcone-4-0-[2"-O-(5'"-O-trans-cinnamoyl)-~­ D-apiofuranosyl]-~- D-glucopyranoside (4), 5,7 -dimethoxy-flavanone-4'-0- [~­ D-apiofuranosyl(I~2)]-~-D-glucopyranoside (5) isolated from ethyl acetate fraction of V. album ssp. album were investigated (Fig 8). The ethyl acetate fraction showed a remarkable and dose-dependent anti-inflammatory activity on carrageenan-induced hind paw oedema model (between 24.7 to 37.2%). Same pharmacological profile was also observed for the compound 2 (30.7-33.6%) from the ethyl acetate fraction. The anti-inflammatory activity of 2 was almost as potent as that of indomethacin which exerts 33.3-42.7% anti-inflammatory activity. Other flavonoids, 1, 3, and 5, isolated from the ethyl acetate fraction, also showed significant but weaker antiinflammatory activity against the same model. Among the isolated compounds from the ethyl acetate fraction were administered in 30 mg! kg dose, compounds 2 and 5 were found to possess highest and significant antinociceptive activity on p-benzoquinone induced writhing reflex test, but not as potent as ASA. The ethyl acetate fraction and isolated compounds did not induce any apparent acute toxicity during the 24 h observation period. It is noteworthy that, in spite of a weak gastric lesion incidence in the higher dose of ethyl acetate fraction (gastric lesions were observed in the stomach of lout of 6 rats), all the isolated flavonoids were found completely safe from the view point of gastric damage (Deliorman et al., 2006). Herencia et al. (1999), investigated the effects of a series of chalcone derivatives on various in vivo and in vitro inflammatory models (i.e. human neutrophil functions, eicosanoid release, TNF -ex production, air-pouch, etc.) and reported inhibitory effects on iNOS and COX-2. Correa et al. (2001) reported potent antinociceptive activity for 3,4-dichlorochalcones using the writhing test in mice. In the study of Pelzer et al. (1998) it was concluded that the anti-inflammatory activity offlavonoids increase depending upon the catechol or guaiacol-like substitution to Bring (3', 4'-dihydroxy or 3"hydroxy-4'-methoxy or 3'-methoxy-4'-hydroxy). Accordingly flavanones such as eriodictyol, 7-0-methyleridictyol and hesperidin showed the highest activity against carrageenan-induced edema. However, flavanone and chalcone derivatives demonstrated to possess anti-inflammatory activity in the present study were shown to possess one glucosylated hydroxyl group at Bring [5, 7-dimethoxy naringenin (1, 3, and 5) or 4', 6'-dimethoxy chalcononaringenin (2, 4) derivatives]. Therefore, results showed herein present additional structural data for the anti-inflammatory

Effects of Medicinal Plants in Turkey

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and antinociceptive activity evaluation of chalcone and flavanone derivatives.

8

°

~9

6

5

~o

4

° (1)

HO HO~OH

14°"f-°----i-CH20H OH

5

°

(2)

°

°

or~/~f HO HO

(4)

Fig 8. Contd.

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Camp. Bio. Nat. Prod., Vol. 2 -Efficacy, Safety & Clinical Evaluation I

Fig 8. Contd.

HO~'''' ~"

I

1 """"-

2

~'HO OH

3' 4'

6

0

1" 0

CH20H

5

3

(5)

Fig 8. Structures of investigated compounds

Arceuthobium oxycedri (DC.) Bieb., is a semi-parasitic plant grows on Juniperus oxycedri and the shape and color ofthe plant looks very similar to host plant. In Anatolia this plant is believed to be effective against a wide range of diseases; from inflammatory to infectious (Yesilada et al., 1995; Fujita et al., 1995; Honda et al., 1996). To confirm the conventional usage, prepared ethanol and aqueous extracts of the plant were studied and both extracts exhibited significant anti-inflammatory effect at the ranges of 28.9-34.2% and 21.1-30.5%, respectively. The plant also showed a potent antinociceptive activity; for the aqueous extract 34.2% at 500 mgt kg dose, while for the ethanol extract 28.6% at 250 mg/kg and 39.9% at 500 mg/kg doses. Results supported the folkloric utilization of the plant as no such study has been carried out on this plant previously (Kupeli et al., 2007c).

LYCOPODIACEAE Lycopodium genus (syn. Huperzia), commonly known as "club moss, ground pine, devil's claw, devil ash" in English, is a pteridophyte abundantly found in subtropical and tropical forests in the world, which is under the risk of extinction (Lawrence, 1989). In Turkey, the genus is represented by five species, namely L. alpinum L., L. annotinum L., L. clavatum L., L. complanatum ssp. chamaecyparissus (A. Br.) Doll, andL. selago L. (Davis & Cullen, 1984). Among them, L. clavatum (LC), and has been reported to be used for wound-healing effect as well as against nappies occurring in babies and, therefore, also called "belly powder" (Baytop, 1999). The spores ofthe plant were stated to have a protective effect as dusting powder for tender